Universal and group‐specific real‐time PCR diagnosis of flavescence dorée (16Sr‐V), bois noir (16Sr‐XII) and apple proliferation (16Sr‐X) phytoplasmas from field‐collected plant hosts and insect vectors

Abstract

Three real-time PCR-based assays for the specific diagnosis of flavescence dorée (FD), bois noir (BN) and apple proliferation (AP) phytoplasmas and a universal one for the detection of phytoplasmas belonging to groups 16Sr-V, 16Sr-X and 16Sr-XII have been developed. Ribosomal-based primers CYS2Fw/Rv and Taq-Man probe CYS2 were used for universal diagnosis in real-time PCR. For groupspecific detection of FD phytoplasma, ribosomal-based primers fAY/rEY, specific for 16Sr-V phytoplasmas, were chosen. For diagnosis of BN and AP phytoplasmas, specific primers were designed on non-ribosomal and nitroreductase DNA sequences, respectively. SYBR Ò Green I detection coupled with melting curve analysis was used in each group-specific protocol. Field-collected grapevines infected with FD and BN phytoplasmas and apple trees infected with AP phytoplasma, together with Scaphoideus titanus, Hyalesthes obsoletus and Cacopsylla melanoneura adults, captured in the same vineyards and orchards, were used as templates in real-time PCR assays. The diagnostic efficiency of each group-specific protocol was compared with well-established detection procedures, based on conventional nested PCR. Universal amplification was obtained in real-time PCR from DNAs of European aster yellows (16Sr-I), elm yellows (16Sr-V), stolbur (16Sr-XII) and AP phytoplasma reference isolates maintained in periwinkles. The same assay detected phytoplasma DNA in all test plants and test insect vectors infected with FD, BN and AP phytoplasmas. Our groupspecific assays detected FD, BN, and AP phytoplasmas with high efficiencies, similar to those obtained with nested PCR and did not amplify phytoplasma DNA of other taxonomic groups. Melting curve analysis was necessary for the correct identification of the specific amplicons generated in the presence of very low target concentrations. Our work shows that real-time PCR methods can sensitively and rapidly detect phytoplasmas at the universal or groupspecific level. This should be useful in developing defence strategies and for quantitative studies of phytoplasma-plant-vector interactions.

Rapid loop-mediated isothermal amplification assays for grapevine yellows phytoplasmas on crude leaf-vein homogenate has the same performance as qPCR

“…Typical symptoms of all GYs include leaf curling and discoloration of leaf veins and lamina, inter-vein yellowing or reddening (according to the variety), uneven or total lack of lignification of canes, flower abortion, and berry withering. Symptoms caused by BNp and FDp are not distinguishable by visual inspection and as the distribution of phytoplasma is uneven within a host that has a very low titre (Prezelj et al 2013), only specific molecular approaches are suitable for the accurate and reliable detection of BNp and FDp. In diagnostic laboratories, assays based on real-time PCR (qPCR) are widely used for the detection of BNp and FDp (Angelini et al 2007;Bianco et al 2004;Galetto et al 2005;Hren et al 2007;Margaria et al 2009;Pelletier et al 2009). Furthermore, a protocol for the detection of FDp based on loop-mediated isothermal amplification (LAMP) assay was developed recently (Notomi et al 2000;Kogovšek et al 2015).…”

Real-time PCR applied to study on plant pathogens: potential applications in diagnosis - a review

“…In addition, an increasing number of real-time PCR procedures are available for detection and quantification of bacteria in plant materials, although TaqMan probes are the most commonly used ones ( Table 2). The lack of growth in pure culture, uneven distribution in the phloem of the infected plant, and low concentration (especially in woody hosts) are most important obstacles for efficient diagnosis of phytoplasmas, that means their quantification can only be achieved in the presence of high levels of host DNA (Galetto et al 2005;Weintraub & Jones 2010). So, a few studies have been done to detect the pathogenic phytoplasmas in plants (Table 2).…”

Polymerase Chain Reaction for Phytoplasmas Detection

“…Phytoplasma pyri' (pear decline, PD), 'Ca. Phytoplasma pruni' (European stone fruit yellows, ESFY) important pathogens of fruit trees (12,76,48,156,3,4,113,23,128,41). Most of the primer/probe systems are targeting 16S rDNA gene though some others genes or even randomly cloned DNA fragments to which no specific function is assigned have been used (Table 3).…”

“…Several laboratories have proposed rapid, specific and sensitive diagnostic protocols for detection of quarantine and economically important phytoplasmas of fruit trees and grapevine such as flavescance dorée (FD) and bois noir (BN) phytoplasmas infecting grapevine (22,48,8,53,71,14); 'Ca. Phytoplasma mali' (apple proliferation, AP), 'Ca.…”

“…For preliminary screening, 16S rDNA gene were adapted for the universal diagnosis of phytoplasmas using direct real-time PCR amplification (30,48,71) (Table 3) and all of them exploited a TaqMan probe for detection. TaqMan probes are labelled at the 5'end with reporter dye and at the 3' end with a quenching molecule; during each PCR cycle in the presence of the specific target DNA, the TaqMan probe, bound to its target sequence, which is then degraded by the 5'-3' exonuclease activity of the Taq polymerase as it extend the primer.…”