Fruit production provides considerable incomes for the growers in many countries worldwide. Stone fruits are extensively grown in most of the EU, although species distribution varies greatly among the Mediterranean and northern European countries (EFSA 2014). This production is particularly significant for Montenegro, where climate provides favorable growing conditions. In the fruit production structure in Montenegro, plum is the most widespread stone fruit species, followed by peach (Prenkić et al. 2016). In this area, stone fruits are grown on intensive and semi-intensive plantations. However, almond production is mainly organized on family gardens and for household consumption. Due to the extensive production and lack of efficient control, Montenegro stone fruit and almond production are often compromised by different phytopathogenic bacteria, especially when environmental conditions favor infection and spread. This population of pathogens was not studied in details recently. Apart from Xanthomonas arboricola pv. pruni detected in some peach samples (Popovic et al. 2020), isolations indicated dominant presence of pseudomonads in the stone fruit samples collected during two growing seasons.
Pss, the causal agent of stone fruit bacterial canker, is one of the 60 pathovars that infects and causes economic losses in many plant species belonging to various families (Young 2010). The pathogen attacks different plant organs of stone fruits and almond causing cankers and necrosis of woody tissue, blight of buds and flowers, spots on the leaves and fruits, and wilting of twigs.
In this study, we characterized bacterial isolates obtained from apricot, peach, nectarine, sweet cherry, Japanese plum, and almond originating from different geographical locations in Montenegro. A total of 29 isolates were identified based on morphological, pathogenic, biochemical and molecular characteristics.
The isolates were isolated during early spring and summer from stone fruits in two subsequent growing seasons. This part of the season was indicated as the most favorable for pathogen isolation (Giovanardi et al. 2018) since the multiplication and spreading of bacteria from active cankers present on fruit trees is the most intense in spring, reaching a maximum during flowering stage (Spotts et al. 2010). The pathogen was isolated from symptomatic plant material—leaf and fruit lesions, branch or twig cankers, and necrotizing buds and petioles. During the 2 year survey of orchards and almond trees, over 100 symptomatic samples were collected, but not all isolations yielded pathogenic bacteria, indicating different disease etiology. Therefore, symptoms could not be a reliable diagnostic criterion, since similar symptoms may be caused by other biotic or abiotic factors. Some fungi also affect stone fruits causing similar symptoms (e.g.,: Wilsonomyces carpophilus, formerly Coryneum beijerinckii causing shot-hole on leaves, twig blights, and bud necrosis): they may be confused with bacterial infections, leading to wrong orchard management. In order to differentiate pathogenic isolates, tobacco hypersensitivity was used as discriminatory test. All tested isolates induced HR in tobacco leaves and produced a fluorescent pigment on KB. Although growth on KB and production of green fluorescent pigment is a common differential characteristic of the genus Pseudomonas, this test can be indicative for the distinction of Pss and Psm from Psp.
In pathogenicity tests, we successfully reproduced the symptoms on inoculated host plant tissues. Obtained results showed that use of the detached organs (shoots, leaves, and fruits) instead of the whole plant is practical and provides reliable results. Additionally, the detached leaf inoculation test has some advantages: symptoms develop quickly, leaves could be used for a longer period than shoots and fruits, the method is rapid, reproducible and simple to perform (Moragrega et al. 2003; Bedford et al. 2003).
Growth and biochemical characteristics of our isolates indicated that they belong to the species P. syringae. Besides positive HR and fluorescence, all the isolates were Gram-negative, catalase positive, metabolized glucose oxidatively, and hydrolyzed aesculin.
Considering the growth on NA and KB plates, an exception was the strain B7, which produced a brown pigment in these media. Such ability of Pseudomonas sp. was reported by Kałużna et al. (2013) and Kałużna (2019) in isolates isolated from cornelian cherry (Cornus mas) and blueberry (Vaccinium corymbosum) in Poland. LOPAT tests have been routinely used to differentiate P. syringae from other fluorescent Pseudomonas species (Lelliott et al. 1966). However, some of the studied isolates did not clearly produce levan, therefore not entirely matching with the LOPAT scheme. Similarly, such behavior was also described for some P. syringae isolates obtained from stone fruits. They formed flat, no levan-type colonies, but still identified as Pss (Roos and Hattingh 1983; Gavrilović 2006).
P. syringae pathovars affecting stone fruits could be differentiated according to another set of differential tests, known as GATTa tests. The results indicated considerable variation among the isolates in these traits (Table 3). The majority of our isolates did not match exactly with the control Pss strain. Aesculin hydrolysis was the only characteristic the isolates had in common, while the other three isolates were variable, indicating limited differential value of GATTa tests in case of our isolates. In addition, the results from the GATTa tests for other P. syringae pathovars are still unknown, which may cause further complications in using these tests.
Gašić et al. (2012) reported that pathovar morsprunorum lost its vitality and showed negative catalase reaction after 4 days on NSA plates, while the pathovars syringae and persicae remained vital for at least seven days. Our isolates maintained their vitality longer than 4 days. Ice nucleation activity (INA) at − 4 °C also indicated close relatedness of the tested isolates to pv. syringae. Some strains of P. syringae, including pv. syringae, catalyze ice crystal formation on and in plant tissues (Lindow 1983). According to Roos and Hattingh (1983), the INA test is quite reliable in differentiating the pv. morsprunorum that shows no INA. However, these authors pointed out that some strains of pv. syringae, as well as intermediate strains, did not show any INA (Roos and Hattingh 1983).
Our experience supports earlier conclusions that biochemical tests are not sufficient and reliable for differentiating P. syringae strains at or below the pathovar level (Little et al. 1998). Biochemical tests, though useful for identification of bacterial genera and species, are often not discriminatory enough to determine P. syringae pathovars (Morris et al. 2000). Berge et al. (2014) reported that phenotypic characteristic provide only limited means for identification of isolates at the clade or phylogroup level and that phenotypes of isolates are variable among and within phylogroups. Pathovars of P. syringae, associated with stone fruits and nuts, produce several well-characterized phytotoxic compounds that can be used for pathovar differentiation. Syringomycin is a low molecular weight non-specific toxin, produced by P. syringae pvs. aptata, atrofaciens, and syringae. This phytotoxin is the main virulence factor of Pss and it is responsible for necrosis development in plant tissues: because of this trait, Pss is distinguished from Psm and Psp. Thus, detection of syrB gene was routinely used for determination of Pss strains (Sorensen et al. 1998; Scortichini et al. 2003; Gilbert et al. 2009; Kałużna et al. 2010). In our study, the syrB gene coding for syringomycin synthesis was detected in 25 tested isolates. Bultreys and Kałużna (2010) also reported the presence of Pss strains negative for syringomycin production. Interestingly, some P. syringae strains may lose this ability when stored for a long period (Hwang et al. 2005).
Our investigation of the Pseudomonas strains collection, by using classical techniques and conventional PCR, was followed by genomic fingerprinting using BOX-PCR and analysis of 16S rRNA gene, gyrB, rpoD, gapA, and gltA genes as phylogenetic markers, leading to the identification of different genomic groups. The results of rep-PCR using the BOX-A1R primer revealed high genetic diversity of the Pseudomonas syringae isolates obtained from stone fruits in Montenegro. The genetic profiles did not support any grouping of the isolates, neither by their biochemical characteristics, nor by their host plant or origin. Abbasi et al. (2013) determined high genetic diversity of Pss isolates obtained from various stone fruit trees in Iran by rep-PCR and IS50-PCR. This study indicated that the rep-PCR technique is reliable, reproducible, and highly discriminative in assessment of the Pss strains diversity (Abbasi et al. 2013). Recent rep-PCR studies have also indicated that pv. morsprunorum strains form a more homogenous group than pv. syringae strains (Vicente and Roberts 2007; Gilbert et al. 2009; Kałużna et al. 2010).
Sequence analysis of the 16S rRNA gene and of housekeeping genes as well as repetitive elements have been described as useful methods for identification and classification and for diversity studies of strains belonging to P. syringae (Bultreys and Kałużna 2010; Hwang et al. 2005). The 16S rRNA gene was analyzed for four isolates, which did not have a gene for syringomycin synthesis. Obtained results showed these isolates belong to Pss.
Multilocus Sequence Analysis (MLSA) method revealed discrimination among P. syringae strains (Sarkar and Guttman 2004; Hwang et al. 2005; Kałużna et al. 2010). It is a recommended method for the determination of genomic relatedness among bacterial strains, where sequences of some housekeeping genes of the bacterial core genome are compared (Sarkar and Guttman 2004). MLSA of 28 representative isolates of P. syringae, isolated from different Prunus spp. and localities in Montenegro demonstrated genomic differences between the isolates that could not be related to the geographic origin or host species. Most of the isolates, with the exception of strain K6, were grouped within genomospecies 1, P. syringae sensu stricto, corresponding to P. syringae PG 2, together with the reference Pss strains. However, the isolates did not form a monophyletic group. They clustered in three separate clades corresponding to previously described PG 2a, 2b, and 2d (Berge et al. 2014). Isolates isolated from apricot, almond, and Japanese plum were distributed in three different clades, while peach and nectarine isolates were included into clade 2b. One isolate from Japanese plum and one from sweet cherry were grouped together with newly described species P. cerasi (Kaluzna et al. 2016). Only strain K6, isolated from apricot, was most distinct from the others and closely related with the reference strains belonging to PG 3, genomospecies 2, which includes stone fruit pathogens Psm race 1 and P. syringae pv. cerasicola as well (Ruinelli et al. 2019). However, it was positive for syr B gene, commonly produced by pathovar syringae; thus, the taxonomic status of this strain within P. syringae species complex should be further studied. Neither of the isolates were closely related to the quarantine peach pathogen Psp, nor Psm race 2 belonging to PG1 (Ruinelli et al. 2019). Based on Ruinelli et al. (2019) genome-based phylogenetic study, isolates obtained from Prunus spp. are distributed throughout PG1, PG2 and PG3 and do not form a monophyletic group within the same PG (Ruinelli et al. 2019). Hulin et al. (2018) reported that isolates of Pss obtained from Prunus were not monophyletic and were found throughout phylogroup 2. PG 2 shows the greatest host diversity, and includes most of the pathovar syringae strains (Hwang et al. 2005).
All tested isolates showed high level of resistance to copper, suggesting resistance development in Montenegrian P. syringae population. This may be due to frequent use of copper compounds in stone fruit and almond disease control programs. Copper resistance in plant pathogenic bacteria is not a rare consequence of an intensive chemical protection in fruit-cultivating regions (Sulikowska and Sobiczewski 2008; Giovanardi et al. 2015, 2017, 2018). On the contrary, bactericidal effect of streptomycin indicated that treatment based on this bactericide could potentially improve control efficacy of the disease. However, antibiotics are not permitted in plant protection in Montenegro.