A variety of pectolytic organisms, referred to as soft rot Pectobacteriaceae (SRP), cause black leg and soft rot of potato (Solanum tuberosum) world-wide (van der Wolf et al., 2021). The diversity of pathogens causing outbreaks is generally regionally specific, although the spread on vegetative materials may mask some regional differences (Toth et al., 2011). The taxonomy of SRP has evolved and during the past four years, a significant number of new Pectobacterium Waldee, 1945 emend. Hauben et al., 1999 and Dickeya Samson et al., 2005 species have been described (Toth et al., 2021; Parte et al., 2020). Although descriptions of new species and taxonomic reviews of these genera provide sequences of individual or multiple genes or whole genomes for comparison, comprehensive comparisons of type strains for diagnostic characters are not available and the resultant identifications generally based solely on sequence analyses (Curland et al., 2021; Motyka-Pomagruk, 2021; Sarfraz et al., 2020, Theron et al., 2022).

The deposition of 16S rRNA sequences is required to describe new species according to various minimum standards to be followed according to the International Code of Nomenclature of Prokaryotes (Parker et al., 2019, Tindall et al., 2010) leading to their use as the most important barcode for species identification for bacteria including members of the SRP (Sławiak et al., 2009). The 16S rRNA locus has been extensively used to characterize the Pectobacteriaceae (Czajkowski, 2009; Raoul des Essarts, 2016; Ma et al., 2007) and to describe new species (van der Wolf, 2014; Gevers, 2005; Khayi et al., 2016, Nabhan et al., 2012). However, 16S rRNA sequence comparisons provide limited taxonomic discrimination at the species level (Ma et al., 2007, Ranjan et al., 2016). Chun et al. (2018) recommended a two-step process starting with 16S rRNA sequence analysis for the taxonomy of prokaryotes. Surveys of SRP have used other single genes as ‘barcodes’ (e.g., gapA, Sarfraz et al., 2020; dnaX, Sławiak et al., 2009), however, databases for these are not as widely available. Although whole genome sequence analysis is the new gold standard for differentiating species, whole genome sequences are not yet required to propose new species and may not be available for all type strains or isolates being evaluated. Additionally, plant disease clinics, the first line of defense in the detection of newly introduced phytopathogens, are not regularly using whole genome sequencing to identify bacterial pathogens (Bull and Koike, 2015). Nevertheless, some SRP surveys have used up to 13 housekeeping genes for MLSA (Sarfraz et al., 2020), but previous studies indicated that the concatenated housekeeping genes gyrB-dnaJ-dnaX are sufficient to distinguish species of Dickeya and Pectobacterium for plant health surveys (Brady et al., 2012, Curland et al., 2021, Marrero et al., 2013, van der Wolf et al., 2014, Khayi et al., 2016, Moretti et al., 2016, Oulghazi et al., 2019).

Likewise, REP-PCR has been used in a variety of studies to differentiate Pectobacterium and Dickeya strains (Czajkowski et al., 2009; Sławiak et al., 2009; Ngadze et al., 2012, Golanowska et al., 2017). However, it is not clear whether comparisons of field isolates to type strains is sufficient for isolate identification especially considering the recent taxonomic splintering of these genera.

Despite the tendency to rely on genetic data for identification, species proposals still require comparative biochemical and physiological data (Parker et al., 2019, Tindall et al., 2010). The methods used for acquiring phenotypic data vary for each nomenclatural proposal. The Biolog GEN III MicroPlate plates have been increasingly used to describe novel Pectobacterium and Dickeya species because of their ease of use and ability to consistently compare a wide array of substrates and traits to differentiate between closely related species (Portier et al., 2019, Waleron et al., 2018, Waleron et al., 2019a, Waleron et al., 2019b, Pédron et al., 2019, Tian et al., 2016). Alternatively, other commercial phenotyping tools such as bioMérieux Biotype assays have been used to define Pectobacterium betavasculorum (Thomson et al., 1981) Gardan et al., 2003, P. wasabiae (Goto and Matsumoto, 1987) Gardan et al., 2003, D. dadantii subsp. dadantii Samson et al., 2005, emend. Brady et al., 2012, D. dianthicola Samson et al., 2005, D. dadantii subsp. dieffenbachiae Samson et al., 2005, emend. Brady et al., 2012, and D. zeae Samson et al., 2005. Given the variations in the methodologies and the depth of phenotype testing carried out, there is no reference that directly compares the phenotypes of members of the SRP, particularly the type strains of Pectobacterium and Dickeya species. This manuscript aims to fill this gap by providing such a reference.

New species of Pectobacterium and Dickeya have become major concerns throughout the U.S. (Curland et al., 2021). Although the origin of these pathogens in the U.S. remains unknown (Johnson, 2015), Dickeya and Pectobacterium species have led to significant yield losses across the northeast since 2014, particularly in Maine (Johnson et al., 2017) and New York (Ma et al., 2018). The 2014 outbreaks sparked interest in monitoring not only endemic pathogens but also for the potential introduction of the highly virulent quarantine pathogen, Dickeya solani van der Wolf et al., 2014. D. solani was found to be associated with severe disease outbreaks in Europe in 2011 (Toth et al., 2011). To date, D. solani has not been identified in the U.S. (Charkowski, 2018), although two other Dickeya species, D. dianthicola Samson et al., 2005 and D. chrysanthemi (Burkholder et al., 1953) Samson et al., 2005, have been isolated from symptomatic potatoes in the U.S. Due to the threat posed by D. solani and other SRP, we sampled potatoes in Pennsylvania to determine the diversity of SRP present on symptomatic potatoes and to begin regional monitoring for introduced potato pathogens.

In this study, we have successfully identified SRP isolated from 2016 to 2018 from symptomatic potatoes in Pennsylvania, employing the aforementioned methodologies. Our study not only offers extensive comparative data for type strains of Pectobacterium and Dickeya species, which can be valuable for future identification efforts, but also marks the first reporting of various SRP species within Pennsylvania and the U.S. Furthermore, it introduces a strategic approach for regional monitoring of emerging SRP strains.