PSA in kiwifruit plants: a new method for Molecular Diagnosis
By a project of Italian Ministry of Agriculture (MIPAAF), the team Phytobacteriology of DAFNE, the University of Viterbo (Italy), after having recently studied in detail the genomes of the main PSA world populations (dx.plos.org/10.1371/journal.pone.0036518), and having clarified the strategies adopted by the bacterium to infect kiwifruit plants and their consequent reactions (see previous article), has now also developed a new diagnostic method for molecular identification of PSA.
The research, entitled: A Multiplex PCR Assay for Detection of Pseudomonas syringae pv. actinidiae and Differentiation of Populations with Different Geographic Origin (Authors: GM Balestra, MC Taratufolo, BA Vinatzer, A Mazzaglia, http://dx.doi.org/10.1094/PDIS-06-12-0590-RE) will soon be published on Plant Disease scientific journal; it has been realized on the basis of a collaboration between the University of Tuscia DAFNE, and the team of the Department of Plant Pathology at Virginia Tech University, USA, coordinated by Prof. Vinatzer.
By the methods developed for the molecular identification of PSA to date, none had yet been able to ensure a complete selectivity respect to pathovar actinidiae and contemporaneously, discriminate between different populations of Pseudomonas syringae pv. actinidiae isolated in different world kiwifruit areas.
Therefore, it was then developed and validated a new assay using multiplex PCR techniques. The assay was tested on 32 isolates of PSA and 15 bacterial strains closely related to PSA. Three different haplotypes of PSA were used as a reference for 3 main groups: a group Japanese/Korean, a European group, and a group of China.
Below: bacterial strains used, their source, geographic origin and host of isolation. Click here to enlarge the table.
Concerning New Zealand strains, 2 virulent strains (PSA V) have been associated to the Chinese group, while, 2 low virulent strains (PSA LV), were negative. The PCR assay has showed a proper level of detection of PSA, equal to 5 - 50 pg of purified DNA (equivalent to 5 x 10² bacteria)/PCR reaction, and has demonstrated its effectiveness both on artificially inoculated plant tissues and on plants naturally infected by PSA.
Multiplex PCR amplification with all four primer pairs (click here to enlarge): Lanes 1-6: Japanese Psa strains (KW1, KW11, 4911, 4912, 5095, 5097), lanes 7-9: Korean Psa strains (Kn2, 23663 ,23664), lanes 10-25: European Psa strains (PSA92, 7285, 7286, 7287, 490, 1To, 15ER, 829, 830, 820, 832, 835, 1F, 5F, 14F, LSV38.17), lanes 26-28 Chinese Psa strains (CH2010-5, CH2010-6, CH2010-7), lanes 29-30: New Zealand Psa strains (18839, 18875); lanes 31-32: New Zealand strains isolated from kiwifruit plants but that do not cause canker (18804, 18882), lanes 33-34: P. syringae pv. theae (2598 and 2599); lane 35: P. syringae pv. tomato; lane 36: P. syringae pv. papulans; lane 37: P. syringae pv. aptata; lane 38: P. syringae pv. lachrymans; lane 39: P. syringae pv. pisi; lane 40-41: P. syringae pv. syringae (3909 b,1 and 4250,1); lanes 42-44: P. avellanae (NCPPB 4224, NCPPB4226, Pav34); lane 45: P. syringae pv. morsprunorum; lane 46: P. syringae pv. maculicola; lane 47: P. viridiflava; lane 48: control-no template DNA. M: molecular marker (GeneRulerTM 100 bp DNA ladder, Genenco). The arrows indicate respectively the PSA amplicon (311 bp), the European amplicon (733 bp), the Japanese/Korean amplicon (609 bp) and the Chinese amplicon (254 bp).
The method described is an innovative tool for the detection/identification of PSA and, particularly practical to verify the geographical origin of the pathogen. This method results therefore particularly useful to test large numbers of vegetative samples both symptomatic and moreover asymptomatic, where PSA may be present. Due to these features, it seems of particular affordability during the surveillance, monitoring and prevention activities by National and Regional Phytosanitary Services as well as by other technical services in charge.
Contact:
Prof. Giorgio M. Balestra
DAFNE (Department for Agriculture, Forestry, Nature and Energy)
University of Tuscia
Via S. Camillo de Lellis
01100 Viterbo - Italy
Tel: +39 (0)761 357474
Mob: +39 333 4246404
Fax: +39 (0)761 357473
Email: balestra@unitus.it
Web: www.unitus.it
Skype: giorba5618
Personal Page: www.agraria.unitus.it/interna.asp?idCat=244