Multiplex PCR for specific and robust detection of Xanthomonas campestris pv. musacearum in pure culture and infected plant material

Abstract

The present study developed a pathovar-specific PCR for the detection of Xanthomonas campestris pv. musacearum (Xcm), the cause of banana xanthomonas wilt, by amplification of a 265-bp region of the gene encoding the general secretion pathway protein D (GspD). A distinct DNA fragment of the expected size was amplified from genomic DNA from all of 12 Xcm isolates tested and no amplification of DNA was observed from other xanthomonads or plant-associated bacteria, including the two closely related species Xanthomonas vasicola pv. holcicola and Xanthomonas axonopodis pv. vasculorum. The Xcm-specific PCR was successfully multiplexed with internal control primers targeting 16S rDNA for application on DNA from bacterial cultures and with primers targeting plant mitochondrial 26S rDNA for application on DNA extracted from plant material. Diagnostic discrimination of healthy and infected plants was subsequently demonstrated in tests on artificially inoculated screenhouse cultivars of banana and field bananas with and without symptoms sampled from different parts of Uganda. This study therefore demonstrated a robust and specific Xcm diagnostic tool with the added advantage of applying internal PCR controls for direct quality assessment of results.

Risks Posed by Intercrops and Weeds as Alternative Hosts to Xanthomonas campestris pv. musacearum in Banana Fields

“…), small plantlets (wild sorghum), or tissue culture plantlets (banana). To rule out any latent Xcm-infection in vegetatively propagated plants, cross sections from the stems and or leaves were sampled, total DNA extracted as described by Mahuku (2004) and checked with PCR using Xcm GspDm-specific primers (Adriko et al, 2012). The plants were then grown in small pots (3 L in size) filled 3/4 full with pre-sterilized forest top soil mixed with sand in a ratio of 2:1 over a period of 1–2 months (depending on the crop species) before treatment application.…”

“…Plates were sealed and incubated at 28°C for a period of 72 h. Single colonies with Xcm–characteristics (yellow, mucoid, and dome shaped) were carefully picked, streaked on fresh media and incubated as above. Resultant colonies were confirmed using Xcm-specific primers (Adriko et al, 2012) using PCR, and a suspension of the bacteria adjusted to 0.5 OD 600 (∼1 × 10 8 colony forming units) using a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc., Pittsburgh, PA, United States) for the inoculation of plants.…”

“…Plates were sealed, incubated at 28°C for 3 days and scored for presence or absence of colonies with Xcm characteristics. All Xcm-like colonies were streaked on fresh YPGA media and incubated as above to obtain pure cultures and confirmed through PCR using Xcm GspDm-specific primers (Adriko et al, 2012). …”

“…The integrity (concentration and purity) of DNA samples was determined using the NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific Inc., Pittsburgh, PA, United States) and adjusted to 50 ng/μL, for PCR. The gDNA extracted from Xcm-like colonies was used as template in a PCR reaction using 265 bp GspDm-specific Xcm primers (Adriko et al, 2012). Amplification reactions were carried out in a 20 μL reaction volume with a final concentration of 0.3 μM of each of the forward and reverse primers, 1.5 mM MgCl 2 , 0.2 μM of each dNTPs (Promega, Madison WI, United States), 1× PCR green buffer, 1 unit of HotStarTaq Plus DNA Polymerase (Qiagen, Canada) and 2 μL of genomic DNA (50 ng/μL).…”

First report of bacterial leaf blight of jute (Corchorus capsularisL.) caused byXanthomonas campestrispv.capsulariiin India

“…13, 1600-1602, http://dx.doi.org/10.1080/03235408.2013 extraction using the QIAGEN DNeasy Blood and Tissue kit. PCR was carried out with Xanthomonas campestris specific primer NZ8F3/NZ85R3 (Adriko et al 2012) which …”