Development of a Magnetic Capture Hybridization Real-Time PCR Assay for Detection of TumorigenicAgrobacterium vitisin Grapevines

Abstract

Agrobacterium vitis, the causal agent of grape crown gall, can have severe economic effects on grape production. The bacterium survives systemically in vines and, therefore, is disseminated in propagation material. We developed an assay for use in indexing programs that is efficient and sensitive for detecting A. vitis in grape tissue. Initially, real-time polymerase chain reaction (PCR) primers specific for diverse tumorigenic strains of A. vitis were developed using the virD2 gene sequence. To overcome the effects of PCR inhibitors present in plant tissue, DNA extraction methods that included magnetic capture hybridization (MCH), immunomagnetic separation (IMS), and extraction with the Mo Bio Powerfood kit were compared. The assays incorporating MCH or IMS followed by real-time PCR were 10,000-fold more sensitive than direct real-time PCR when tested using boiled bacterial cell suspensions, with detection thresholds of 10(1) CFU/ml compared with 10(5) CFU/ml. DNA extraction with the Powerfood DNA extraction kit was 10-fold more sensitive than direct real-time PCR, with a detection threshold of 10(4) CFU/ml. All three assays were able to detect A. vitis in healthy-appearing grapevine cuttings taken from infected vines.

Grapevine (Vitis vinifera) Crown Galls Host Distinct Microbiota

“…After 2 h at 28°C, the supernatant was used for 10-fold serial dilutions, and 100 l was incubated for 5 (52) served for differentiation between Agrobacterium vitis and other Agrobacterium species, and (iii) a fragment of the VirD2 was PCR amplified to screen for the presence of the Ti plasmid (11). The following primer sequences were used: (i) 16S rRNA gene primers 27F (5=-AGR GTT YGA TYM TGG CTG AG-3=) and 1492R (5=-GGY TAC CTT GTT ACG ACT T-3=), or 515F (5=-GTG YCA GCM GCC GCG GTA A-3=) and 806R (5=-GGA CTA CNV GGG TWT CTA AT-3=), (ii) Agrobacterium species-specific RecA primers F8360 (5=-AGC TCG GTT CCA ATG AAA-3=) and F8361 (5=-GCT TGC GCA GCG CCT GGC T-3=), A. vitis-specific RecA primers G0004F (5=-GAT ATC GCG CTC GGC ATT GGT-3=) and G0005R (5=-CCT TCG ATT TCA GCT TTC G-3=) (52), and (iii) virD2 primers virD2F (5=-TTG GAA TAT CTG TCC CGG AAG-3=) and virD2R (5=-CTT GTA CCA GCA GGG AAG CTT A-3=) (11). A 50-l PCR mixture contained 1ϫ HF buffer (New England BioLabs, Ipswich, MA, USA), an experimentally determined amount of custom-made Phusion polymerase (53), 0.2 M each primer, 400 M dinucleoside triphosphates (dNTPs) (Fermentas, Waltham, MA, USA), and 2 l of a bacterial colony resuspended and boiled in 100 l of HPLC-grade water (RotisolV HPLC gradient grade; Roth) for 10 min.…”

“…A. vitis is known to persist in debris from infested grapevine material in soil (5) and can enter grapevines via the root and move through the xylem (6) to wounded parts of the plant, where it transforms the cells (7)(8)(9). The pathogen has been detected in the xylem sap of canes, so propagation material of grapevine nurseries serves as an additional risk for distributing A. vitis (10,11). Virulent A. vitis strains harbor a tumor-inducing (Ti) plasmid, which enables the transfer of transfer DNA (T-DNA) into the plant genome, a process supported by virulence (Vir) genes of the Ti plasmid (12,13).…”

Rapid and sensitive methods for detection of Allorhizobium vitis, causal agent of grapevine crown gall

“…Therefore, the disease spreads out by asymptomatic propagating materials (Kuzmanovic et al, 2014) which is necessary to develop a reliable detection methods of the pathogen in both symptomatic and asymptomatic plant materials and in the soil to efficiently prevent the disease (Bini et al, 2008). Generally, the detection methods of A. vitis are based on their isolation on the semiselective culture media and pathogenicity tests but these methods can take many weeks for results (Johnson et al, 2013, Shams et al, 2012. Polymerase chain reaction (PCR) it's the most reliable technique improve sensitivity, specificity and rapidity for the detection of bacteria which targets gens that is found only in A. vitis by the use of specific primers (Johnson et al, 2013;Shams et al, 2012Shams et al, , 2013.…”

“…Generally, the detection methods of A. vitis are based on their isolation on the semiselective culture media and pathogenicity tests but these methods can take many weeks for results (Johnson et al, 2013, Shams et al, 2012. Polymerase chain reaction (PCR) it's the most reliable technique improve sensitivity, specificity and rapidity for the detection of bacteria which targets gens that is found only in A. vitis by the use of specific primers (Johnson et al, 2013;Shams et al, 2012Shams et al, , 2013. Several PCR protocols have been developed and successfully used for detection of tumorigenic strains of A. vitis directly from plant hostswith the use of primers, which target gens localized on Ti plasmid,vir or TDNAregions (Bini et al, 2008).…”

The Impacts of Tumorigenic and NontumorigenicAgrobacterium vitisStrains on Graft Strength and Growth of Grapevines

“…Grafting of grapevines provides a significant wound site at which A. vitis can initiate infection. We previously developed a highly sensitive method for detecting tumorigenic A. vitis in grapevines (Johnson et al 2013). The pathogen was detected in all parts of the vines, including nodes, internodes, shoot tips, and leaves (Johnson et al 2016;Orel et al 2017).…”