A Multiplex PCR Assay for Detection ofPseudomonas syringaepv.actinidiaeand Differentiation of Populations with Different Geographic Origin

Abstract

Pseudomonas syringae pv. actinidiae is responsible for severe outbreaks of bacterial canker of kiwifruit currently occurring around the world. Although molecular detection methods have been reported, none provide complete selectivity for this pathovar or discriminate among pathogen haplotypes. Therefore, a new multiplex polymerase chain reaction (PCR) assay was developed and validated. The assay was tested on 32 P. syringae pv. actinidiae isolates and 15 non-P. syringae pv. actinidiae strains and correctly assigned P. syringae pv. actinidiae strains to three different haplotypes: a Japanese/Korean group, a European group, and a Chinese group. Two P. syringae pv. actinidiae isolates from New Zealand were found to belong to the Chinese group whereas two other isolates from New Zealand, which were isolated from kiwifruit plants but which do not cause bacterial canker, tested negative. The described PCR assays has a limit of detection of approximately 5 to 50 pg of purified DNA or of 5 × 102 bacteria/PCR and were shown to work with both artificially and naturally infected plant tissues. Thus, the described method represents a suitable tool for detection of P. syringae pv. actinidiae and haplotype attribution, in particular, when testing a high number of samples during surveillance and prevention activities.

PCR Multiplexes DiscriminateFusariumSymbionts of InvasiveEuwallaceaAmbrosia Beetles that Inflict Damage on Numerous Tree Species Throughout the United States

“…The newly designed primer sets amplify their correct targets in concentrations at or below 1 ng/ml for all gDNA templates, and at or below 10 pg/ml for three AFC species, which is comparable with previously reported sensitivity for multiplex assays of phytopathogenic fungi (Dreaden et al 2012;Rigotti et al 2002) and bacteria (Balestra et al 2013). The multiplex assays successfully identified AF-3 and AF-4 from pooled DNA from eight batches of freshly macerated E. interjectus and E. validus beetle heads, respectively.…”

Genomic Analysis of the Kiwifruit Pathogen Pseudomonas syringae pv. actinidiae Provides Insight into the Origins of an Emergent Plant Disease

“…The NZ V-13 ICE is also present in the genome of the Chinese outbreak strain C-1, however, as previously indicated [22], [29], this region is highly divergent in the four Italian and two Chilean outbreak strains [27]. A comparison of the Italian (Mediterranean Island), Chilean (Andean Island) and the Pacific Island is depicted in Figure 6.…”

“…The origin of the current global epidemic is the subject of interest and speculation, with reports that China is the likely source [22], [27], [29]. Previous studies identified six SNPs that distinguished European from Chinese isolates [22]; upon inclusion of two Chilean isolates, nine diagnostic SNPs were identified [27].…”

“…A genomic island with similarity to PPHGI from Pseudomonas syringae pv. phaseolicola ( Pph ) was characterized and shown to differ between the European, Chilean and Chinese/New Zealand isolates [22], [27], [29]. The full extent of the genetic distinctiveness of Psa -V relative to strains from early outbreaks remains uncertain.…”

Identification of Pseudomonas syringae pv.actinidiae strains causing bacterial canker of kiwifruit in the Anhui Province of China, and determination of their streptomycin sensitivities

“…Historically considered a minor disease, P. syringae pv. actinidiae rarely caused severe damage to kiwifruit until 2008, when a large outbreak occurred in Italy, and severe infections continue to spread to other countries (Mazzaglia et al, 2011;Chapman et al, 2012;Balestra et al, 2009Balestra et al, , 2013; Vanneste et al, 2013). In Italy, the bacterium has been shown to cause severe disease symptoms, including leaf wilting; twig or cane dieback; cankers on canes, leaders, and trunks; plant wilting; and the production of an oozing red or white exudate (Ferrante and Scortichini, 2010;Bull et al, 2011).…”