Introduction

Walnut species (Juglans sp.) are important nut and timber producer’s intemperate regions of Europe, Asia and North America. The Persian walnut (Juglans regia) is the most horticultural developed and widely cultivated and is easily the leading producer of commercial nuts (Hajri et al. 2010). Therefore diseases affecting this plant will cause important economic losses worldwide. Here we report a spot disease characterized by the formation of necrotic zones or halos in leaves of walnut seedlings in Iran that resembles those produced in other plants by Pseudomonas syringae pv syringae.

The phytopathogenic Pseudomonads cause numerous plant diseases with diverse symptoms including cankers, diebacks, blossom, twig, leaf or kernel blights, leaf spots (Pseudomonas syringae pathovars), soft or brown rots (P. viridiflava and P. marginalis pathovars), tumors or galls (P. savastanoi pathovars), and mushroom blights (P. tolaasii and P. agarici) (Schaad et al. 2001).

P. syringae is a Gram-negative bacterium belonging to the genus Pseudomonas sensu stricto, included in the γ subclass of the Proteobacteria (Kersters et al. 1996). Currently P. syringae divided into more than 50 pathovars and nine genomic species (Bradbury 1986; Gardan et al. 1999). This bacterium has a wide host range as it causes diseases to over 180 plants.

Pseudomonas syringae pv. syringae is a particular bacterium among P. syringae pathovars due to its capacity to cause disease in many species of plants (Little et al. 1998). Traditionally, strains of Pss are recognized based on biochemical, nutritional, and physiological characteristics and ability of pathogenicity on lilac and peach seedling (Little et al. 1998; Scortichini et al. 2002; Vicente and Roberts 2003, 2007; Gilbert et al. 2009, 2010). The Pss host specificity among the strains that infect different hosts such as beans, grasses, and Prunus species were reported on the basis of pathogenicity tests (Little et al. 1998). Many researchers have found that peach seedlings are sensitive to Pss strains from different hosts (Otta and English 1971; Vicente and Roberts 2007; Gilbert et al. 2010).

In Iran, phytopatogenic Pss strains were isolated from stone fruit trees and other plants in different areas which presented different phenotypic and genotypic characteristics among those isolated from various hosts (Bahar et al. 1982; Banapour et al. 1990; Ashorpour et al. 2008; Abbasi et al. 2013).

During the past decade molecular techniques for identification of plant pathogenic bacteria were developed (Louws et al. 1999). Genomic fingerprinting methods based on the polymerase chain reaction (PCR) have been applied for identification and classification of plant associated bacteria at the subspecies level (Louws et al. 1995, 1999). Repetitive extragenic palindromic (REP) sequences or elements were first described in Escherichia coli and Salmonella typhimurium operons (Higgins et al. 1982). Repetitive sequences present in the genomes of diverse bacterial species have been used to design PCR primers that generate reproducible fingerprints useful to assess bacterial diversity at the strain and pathovar level (Versalovic et al. 1991, 1994; Louws et al. 1994). The researchers concluded that these methods could be used to identify and classify strains into different pathovars within Pseudomonas syringae (Weingart and Volksch 1997). Gutiérrez-Barranquero et al. (2008) and Qing et al. (2011) reported that rep-PCR is an interesting tool for the delineation or genotyping of bacterial species because it is more discriminatory than other DNA fingerprinting techniques. Weingart and Volksch (1997); Vicente and Roberts (2007); Gilbert et al. (2009) and Abbasi et al. (2013) by studying the genetic diversity of Pss strains of stone fruits, found that rep-PCR is a rapid and simple method to evaluate of genetic diversity of Pss strains and this method also can assist in the identification of Pss isolates.

The analysis of 16S rRNA gene sequences has become the standard technique for identification of bacteria (Schaad et al. 2001) because it is highly conserved, but present variations among genera and species of prokaryotes which makes this gene very useful for bacterial identification at genus and species level. Nevertheless, other less conserved genes are more useful to differentiate among phylogenetically close bacterial species.

Phylogenetic relationships of Pseudomonads resolved by using gyrB (DNA gyrase B subunit) and rpoD (σ70 factor) sequences were eminently different from those resolved by using 16S rRNA sequences. DNA gyrase is the enzyme responsible for introducing negative supercoils into bacterial chromosomes and plays a crucial role in the replication of chromosomes (Watt and Hickson 1994). The σ70 factor, on the other hand, is one of the sigma factors that confer promoter-specific transcription initiation on RNA polymerase (Lonetto et al. 1992).

Yamamoto et al. (2000) reported that the phylogenetic analysis using the gyrB and rpoD sequences may fill the resolution gap between 16S rRNA sequence analysis and DNA ± DNA hybridization studies.

In this study we isolated for the first time the bacterial strains producing necrotic halos in the leaves of walnut seedlings in Iran. They were characterized by using phenotypic and genetic methods and were identified as Pseudomonas syringae pv. syringae (Pss) by 16S rRNA, gyrB and rpoD gene sequencing.

Materials and methods

Sampling and isolation of the causal pathogen

During 2012, walnut leave samples shown necrotic lesions were collected from different areas in Hamedan province. Plant samples were washed under tap water, disinfected, washed again with sterilized distilled water, cut into small pieces placed in sterilized distilled water for 30 min. A loopful of suspension was streaked on NA medium and put at 25 °C for 3 days. Each colony was streaked onto NA for to isolate pure single colonies. Purified colonies were streaked on Kings’ B medium to detection of fluorescent pigmentation (Hildebrand et al. 1988). The isolates were stored in 30 % (v/v) glycerol solution at −80 °C for long-term storage.

Pathogenicity test

Selected isolates were grown on nutrient agar for 48 h at 27 °C. Leaves of walnut seedling were first surface sterilized with 70 % ethanol and rinsed in distilled sterilized water; then, strains were inoculated by depositing 1 μl of a bacterial suspension (108 CFU/ml) into leaves with sterilized hypodermic needles. Plants were covered with a plastic bag for 24 h to increase relative humidity and were maintained in a greenhouse at 20–25 °C until the symptoms appeared (Schaad et al. 2001). Also, one ml of bacterial suspensions was injected into the green stems of peach and plum seedlings by using a needle (Little et al. 1998). Each plant was inoculated in ten sites with one strain. Sterilized distilled water was used to inoculate plants that were used as negative controls.

Phenotypic features

Phenotypic features of the bacterial strains were characterized based on standard bacteriological methods. These include; Gram staining and sensitivity to 3 % KOH (Suslow et al. 1982), aerobic growth (Hugh and Leifson 1953) and fluorescent pigment production on King’s B medium (Hildebrand et al. 1988). Levan formation, oxidase reaction, potato rot (pectolytic activity), argenine dihydrolase and tobacco hypersensitivity (LOPAT) test was analysed as was previously described (Lelliott et al. 1966). Hydrolysis of gelatin, aesculin and starch, catalase and tyrosinase activities and growth on 4 % NaCl was performed as described by Hildebrand et al. (1988) and growth at 37 °C according to Fahy and Hayward (1983). In addition, utilization of carbon sources was tested on the basal medium of Ayers et al. (1919) supplemented with 0.1 % carbohydrates and amino acids.

DNA preparation

Bacteria were grown on nutrient agar at 27 °C for 48 h. Bacterial suspensions were prepared in sterile distilled water (108 CFU/ml) and were lysed by the addition of 1:10 volume of 3 % KOH and heating the suspension at 95 °C for 2 min with subsequent cooling on ice. Lysates were centrifuged at 8000×g for 2 min, and the supernatants were used directly for PCR or stored at −20 °C until used (Rouhrazi and Rahimian 2012).

PCR assays using rep-PCR

Amplification reaction was carried out using the total DNA extracted from each isolates as template for ERIC and BOX-PCR. Two specific primers were used to correlate to ERIC sequence (Versalovic et al. 1991). ERIC 1R (5′- ATGTAAGCTCCTGGGGATTCA- 3′) and ERIC2 (5′-AAGTAAGTGACTGGGGTGAGC- 3′) (Louws et al. 1994). Also repetitive sequence based PCR typing with the BOX-A1R primer (5′-CTA CGGCAAGGCGACGCTGACG- 3′) (Renick et al. 2008) was carried out as follows. ERIC and BOX-PCR were performed in 25 μl reaction mixtures containing 2.5 μlof 10X PCR buffer, 2.5 mM MgCl2, 0.4 mM dNTPs, 0.2 μlM of each primer, 1.25 U Taq DNA polymerase and 3 μl of template DNA. Amplification was carried out as follows: after initial denaturation for 3 min at 94 °C, 35 amplification cycles were completed, each consisting of 1 min at 94 °C, 1 min at 50 °C (55 °C in BOX-PCR) and 2 min at 72 °C. A final extension of 10 min at 72 °C was applied (Rademaker and de Bruijn 1997). DNA bands obtained by Eric and Box-PCR and subsequently electrophoresed were scored as present (1) or absent (0) and their fingerprints were compared using NTSYS software. Jaccard’s coefficient of similarity index was used to calculate similarity distances. Cluster analysis was done using the un-weighted pair-group method with arithmetic average (UPGMA) (Sneath and Sokal 1973).

Sequence analyses of 16S rRNA, rpoD and gyrB genes

The amplification and sequencing of 16S rRNA, rpoD and gyrB genes were carried out as 164 described previously (Manceau and Horvais 1997; Sarkar and Guttman 2004). The sequences 165 obtained were compared with those from the GenBank using the BLASTN program (Altschul et al. 1990). The obtained sequences and those of related bacteria obtained from GenBank 167 were aligned using the Clustal W program (Thompson et al. 1997). The distances were 168 calculated according to Kimura’s two-parameter model (Kimura 1980). The phylogenetic 169 trees were inferred using the neighbour joining model (Saitou and Nei 1987) and MEGA5.0170 (Tamura et al. 2011) was used for all the phylogenetic analyses.

Results

Isolation and identification of the causal pathogen

From walnut leaf spots (Fig. 1), a total of 46 strains with beige round and smooth margins colonies were isolated on NA medium (Table 1). The isolates were Gram negative, obligate aerobic, and produced fluorescent pigment on King’s B medium. They formed levan from sucrose and were oxidase and arginine dihydrolase negative (Table 2). They were potato rot negative and caused hypersensitivity reaction on tobacco and geranium (LOPAT group Ia). Table 2 showed phenotypic properties of the isolates. The isolates induced leaf spots under greenhouse seven days after inoculation and also natural conditions. They were pathogenic on peach and plum seedling and produced progressive necrotic symptom on the inoculated sites of the stems.

Fig. 1
figure 1

Leaf spot symptoms on walnut seedlings caused by Psudomonas syringae pv. syringae in Iran

Full size image
Table 1 Bacterial isolates used in this study
Full size table
Table 2 Phenotypic features of strains of Pseudomonas syringae pv. syringae isolated from walnut seedling leaf spot in Iran
Full size table

The biochemical, physiological and pathogenicity characteristics indicated that the isolates belong to Pseudomonas syringae pv. syringae.

ERIC-PCR and BOX-PCR analysis

Banding patterns of strains were generated using the BOXA1R and ERIC primers. The bands amplified in the BOX-PCR ranged in length from 250 to 3500 bp (Fig. 2) and those amplified in the ERIC-PCR ranged from 150 to 1500 bp (Fig. 3). Reproducible fingerprint profiles were generated with each technique upon repetition of the procedures. Walnut isolates were differentiated into two clusters at the 90 % similarity level. Group 1 include AK1, AK4, AK5, and AK7 isolated from Tuyserkan and group 2 include AK10, AK13, AK14, AK16, AK19 and AK20 isolated from Hamedan.

Fig. 2
figure 2

PCR band pattern using BOXA1 primer for the strains of Pseudomonas syringae pv. syringae causing walnut seedling leaf spot in Iran. M: 1 kb DNA ladder

Full size image
Fig. 3
figure 3

PCR band pattern using ERIC1/ ERIC2 primers for the strains of Pseudomonas syringae pv. syringae causing walnut seedling leaf spot in Iran. M: 1 kb DNA ladder

Full size image

16S rRNA, rpoD and gyrB genes analysis

Based on the analysis of ERIC- and BOX-PCR fingerprints, the isolates (AK1 and AK10) were selected and alignment of their 1400-bp 16S rRNA sequence determined. The sequences have been deposited in the GenBank database under the accession numbers KU321601 and KU321602. Homology searches with the 16S rRNA sequences in the GenBank database showed that the Iranian isolates are 99.5 similar to Pseudomonas syringae pv. syringae.

The phylogenetic analysis of the 16S rRNA gene sequences of isolated strains within the phylogenetic groups to which belong their closest related pathovars within Pseudomonas syringae are shown in Fig. 4.

Fig. 4
figure 4

Neighbor-joining tree based on the 16S rRNA sequences, showing the relationships between the representative walnut isolates from Iran (filled circles) and different pathovars of Pseudomonas syringae strains (alignment length 1350 bp). Percentage bootstrap is indicated on the internal branches (replicates)

Full size image

The nucleotide sequences of 530 bp ropD and 550 bp gyrB genes were obtained for the representative strains. The analysis of these two genes showed that the strains were phylogenetically close to P. syringae pv. syringae (Fig. 5).

Fig. 5
figure 5

Neighbour-joining phylogenetic tree based on concatenated rpoD and gyrB gene sequences of the strains isolated in this study and different pathovars of Pseudomonas syringae

Full size image

Discussion

The two most common major species of walnuts are grown for their seeds, the Persian or English walnut and the black walnut. The English walnut (J. regia) originated in Persia and the black walnut (J. nigra) is native to North America. The purpose of this study was to isolate the bacterium causing leaf spot of walnut (J. regia) seedling in Iran and to identify it through the analysis of several biochemical and molecular tests and to confirm its pathogenicity in different plants. In this study, the strains of causal agent for leaf spot of walnut presented the same phenotypic characteristics (Table 2) and after the application of a set of tests known as LOPAT (Lelliott et al. 1966) for fluorescent Pseudomonas group differentiation, the isolates present the typical characteristics of P. syringae (formed levan, oxidase and arginine dihydrolase were negative, caused hypersensitivity reaction on tobacco but they were unable to rotten potato slices). Peix et al. (2009) reported phenotypic characteristics such as carbon sources consumption, ability to grow in different culture conditions, antibiotic resistance, producing antibiotics, extracellular enzymes, cell shape and type of flagellum as an easy way to differentiate species from genus Pseudomonas. Nevertheless, Lelliott et al. (1966) had showed that among the fifteen tests to determine and differentiate the fluorescent plant pathogenic Pseudomonas spp. only a set of tests named as LOPAT could differentiate five distinct pathogenic species groups. Bultreys and Kaluzna (2010) also used LOPAT tests to differentiate of fluorescent plant pathogenic Pseudomonas spp. In this study, strains of causal agent for leaf spot of walnut are similar to Pss in LOPAT test and consumption of carbon resources (Table 2).

Scortichini et al. (2002) showed that P. avellanae and strains similar to Pss which are agents of hazelnut decline in northern Greece and central Italy concluded that strains of Pss related to hazelnut in LOPAT test are like P. syringe but in the consumption of carbon resources such as DL-homoserine and tyrosinase differed from P. syringe and have the ability to use these compounds. The authors indicated that the studied strains were a distinct taxon very closely related to Pss.

ERIC and BOX profiles have been used successfully to characterize a large number of bacteria and they differentiate among closely related strains (Versalovic et al. 1991, 1994). These fingerprinting techniques generated characteristic profiles that can be used to differentiate pathogen isolates of Pseudomonas from different species and pathovars and even from the same pathovar (Weingart and Volksch 1997; Little et al. 1998; Joana et al. 2007; Marques et al. 2008; Gašić et al. 2012; Kaluzna et al. 2010; Çepni and Gürel 2012; Abbasi et al. 2013). In this work we confirmed that strains from the same pathovar are differentiated by ERIC and BOX fingerprinting technique since different patterns were obtained for strains causing walnut seedling leaf spot isolated in Hamedan province and strains isolated from Tuyserkan, while the strains of the same region showed identical pattern. All these strains showed 99.5 % similarity to Pss after the 16S rRNA gene analysis in agreement with the results of the identification based on phenotypic characteristics. Nevertheless, several species of genus Pseudomonas have closely related16S rRNA gene sequences and then the analysis of other genes, including gyrB and rpoD, have been used to study genetic diversity and evolution in P. syringae sensu lato and to identify pathovars and strains within this species (Yamamoto et al. 2000; Hwang et al. 2005; Ferrante and Scortichini 2010; Kaluzna et al. 2010; Bull et al. 2011; Martín-Sanz et al. 2012, 2013). The analysis of these two genes in the strains from this study showed that the bacterium causing walnut leaf spot is very close to Pss. To the best of our knowledge, this is the first report of Pseudmonas syringae pv. syringae as the causal agent of walnut seedlings leaf spot in Iran and worldwide.