Potato blackleg and soft rot are the most serious bacterial diseases that inflict significant economic damage to the potato crop worldwide. Various species and subspecies of Pectobacterium spp. and Dickeya spp. (Enterobacteriaceae) are responsible for the outbreaks of these diseases. For a long time, P.atrosepticum (Pba) and P.carotovorum subsp. carotovorum (Pcc) have been identified as the causal agents of blackleg and potato soft rot; however, the recent emergence and presence of some bacteria including D.solani, P. carotovorum subsp brasiliense (Pcb), and P.parmentieri (Pp) in potato fields around the world demonstrated that other Pectobacterium species and subspecies can be involved in the occurrence of potato blackleg and soft rot. These changes most likely related to the global temperature rise and extensive genetic exchange among Pectobacteria. For years, Pba and Pcc have been known as the primary causes of blackleg and soft rot of potatoes in Iran. However, the later detection of D. chrysanthemi (Dch), D. solani, and Pp in the potato growing area of Isfahan province, led to presume that other species and subspecies of Pectobacteria might also spread in the region. These recent pathogens have been likely imported into Iran via the importation of seed tubers from Europe and adapted to the warm condition of potato growing season in Isfahan. To investigate the distribution status of Pectobacteria in Isfahan province, several field surveys were carried out in 2015-2018 to collect potato plants showing symptoms of blackleg and soft rot. Upon isolating bacteria from the samples, the isolates belonging to Pectobactrium spp. were categorized into four groups according on their morphological, physiological, biochemical characteristics and also DNA fingerprinting patterns with BOXIR and ERIC1R/ERIC1F primers. Polymerase chain reaction (PCR) assays using species-specific primer pairs in combination with the nucleotide sequence analysis of the amplified fragments were employed to identify of the isolates at species level. Accrdingly, four Pba, 12 Pcb, 67 Pcc and 110 Pp isolates were identified among the 202 isolates tested; however, none of them belonged to the Dickeya genus. The rare incidence of Pba and preva‎lence of Pcb, Pcc, and Pp as causes of blackleg and potato soft rot in temperate and warm regions of Isfahan was consistent with expectations. The pathogenicity of the isolates was confirmed by testing the representatives of each group under lab and greenhouse conditions. Moreover, the potato slices rot test revealed that the isolates have distinct pectolytic activity at 4°C, 10°C, 20°C, 27°C and 37°C. Due to lack of a certain primer pair for specific targeting of Pp isolates, a unique intergenic region of 3036 bp in length in Pp (isolate WPP161) was employed to develop a species-specific primer for Pp. The specificity and sensitivity of the primer pair for detection of Pp isolates were established through a series of PCR assays and nucleotide sequencing analysis of the amplified fragment. With the goal of selecting a reliable method for monitoring of Pba as a quarantine pathogen, the relative efficiency of three methods including PCR, enzyme-linked immunosorbent-assay (ELISA), loop-mediated isothermal amplification (LAMP) and antibody-based biosensor were eva‎luated in present study. Based on the results, LAMP effectively detected Pba at the concentration of 102 CFU/ml. However, due to the cross reactivity responses of the polyclonal antibodies produced against Pba in this study and its gamma globulin (IgG) extract with other Pectobacteria, the specific detection of Pba through indirect ELISA test was rated unsuccessful. In this study, we report on the optimization of an alternative approach based on an electrochemical impedance immunosensor combining a microfluidic module and a microelectrodes array, and having advantages in terms of low cost, ease of use and portability........

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Giuseppe Maruccio , Palmiro Poltronieri

بهرام شريف نبي , صبيحه سليمانيان زاد , بهرام نصر اصفهاني