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Bacterial canker of olives caused by of Pseudomonas syringae pv. syringae in Iran
Scientia Horticulturae 118 (2008) 128–131
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Bacterial canker of olives caused by of Pseudomonas syringae pv. syringae in Iran Maesomeh Ashorpour a, Mostafa Niknejad Kazempour a,*, Mohamad Ramezanie b a b
Department of Plant Pathology, Faculty of Agriculture, University of Guilan, Rasht Iran Natural Resources of Research Guilan province Rasht Iran
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 February 2008 Received in revised form 3 May 2008 Accepted 27 May 2008
Guilan is one of the main province olive oil producers in Iran. The culture of olive trees is of considerable importance. One problem that affects Iran olive orchards is a disease bacterial canker caused by Pseudomonas syringae pv. syringae. In this research, the bacterium was recovered from sunken brown stem lesions on 2-year-old olive trees cv. Marie, during survey from olive orchards in different areas of Guilan province. Samples were taken from infected tissues and were washed with sterile distilled water and crushed in peptone water. Then 50 ml of the extract was cultured on NA and King’s B medium containing cyclohexamid antibiotic (50 mg/ml). After 48–72 h, bacterial colonies were selected. Based on morphological, physiological, biochemical, pathogenicity properties and PCR methods with specific primers the predominant pathogenic type was identified as P. s. pv. syringae. This is the first report of the existence of P. s. pv. syringae on olive trees in Iran. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Bacterial canker of olive Pseudomonas syringae pv. syringae
1. Introduction Olea europaea L. is one of the oldest agricultural tree crops which is cultivated in about 71 thousand ha, situated in the north, southwest, central west and southeast regions. In recent years, molecular markers have been developed in olives. They include randomly amplified polymorphic DNA (RAPD) (Mekuria et al., 1999). The olive tree is affected by some pests and diseases, although it has fewer problems than most fruit trees. Because the olive has fewer natural enemies than other crops, and because the oil in olives retains the odor of chemical treatments, the olive is one of the least sprayed crops (Ferguson et al., 1994). Threshold temperatures varied between 5 and 12.5 8C depending on biogeographical characteristics (Gala´n et al., 2005). Bacterial stem cankers and dieback of olive caused by Pseudomonas syringae pv. syringae were likely to have entered plants through pruning wounds or where frost/cold injury had caused stem tissue to crack or peel. Symptoms vary from slow decline of trees and tree death, to localized cankers around wound sites (Hall et al., 2003). P. syringae has been reported on olives from Italy, where it has been found epiphytically on olive leaves (Ercolani, 1991) and infecting
* Corresponding author at: Department of Plant Pathology, Faculty of Agriculture, Guilan University, P.O. Box 41635-1314, Rasht, Iran. E-mail address: [email protected] (M.N. Kazempour). 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.05.035
frost damaged plants (Iannotta et al., 1999). In Southern Italy, it was found in association with necrosis and stem girdling at the collar of young trees cv. Carolea, and was attributed to a nutritional imbalance, which weakened the trees and made them susceptible to infection (Scortichini, 1997). The objectives of the present research were the isolation of the causal agent of bacterial canker on olives in the Guilan province and the identification of isolates by biochemical, nutritional, pathogenicity and PCR methods. 2. Materials and methods 2.1. Bacterial isolation Samples were collected from olive orchards in the counties Roudbar, Aliabad, Manjil and Loshan which are located in the north of Iran and have average temperature 22–25 8C and relative humidity 90–95%. Small tissue pieces from stem lesion margins, surfaces of cankers and leaf tissue showing necrotic lesions and blight symptoms were removed aseptically, ground in bacteriological saline (0.85%, w/v, NaCl), and left at room temperature (20 8C) for 10 min. Loopfuls of the bacterial suspension were streaked onto Nutrient Agar (NA) and King’s medium B (KB) and incubated at 26 8C. Pure colonies of bacteria were isolated from the lesions. Isolates were routinely grown on KB at 26 8C and stored at 4 8C for up to 2 weeks. For longer-term storage bacterial isolates were stored in freezing medium at 80 8C.
M. Ashorpour et al. / Scientia Horticulturae 118 (2008) 128–131
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2.2. Pathogenicity test on stem of olive
2.6. PCR conditions for amplification and electrophoresis
Pathogenicity was tested according to Scortichini (1997). Pathogenicity tests were conducted on young potted 18-monthold olive trees cvs. Marie, Kroniky, Conservalia and Arbkin, using 18 isolates of the olive bacterium. Stems were wounded with a 25G hypodermic needle, the wound covered with cotton wool dipped in a suspension of 1 108 bacteria/ml and the inoculation site was wrapped with grafting tape. Two isolates of P. syringae pv. syringae from cherry (DAR 30499 and 33426) were also tested for pathogenicity. A water-only inoculation was included as the control. The trees were kept in high humidity for 24 h before, and after, inoculation when the grafting tape and cotton wool were removed. The plants were then moved outdoors where they were watered overhead twice daily.
Amplification was carried out in a 25 ml volume in 0.5 ml microtube using a Hybaid programmable thermal controller. Each 25 ml PCR reaction mixture contained 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2, 200 mM of each nucleotide (dATP, dCTP, dGTP, and dTTP), 0.25 mM of each primer, 100 ng DNA and 1 U of Taq DNA polymerase (Promega Corp., Madison, WI). A 25 ml sterile, mineral-oil overlay was added to reduce evaporation. DNA amplification was carried out in a PTC-100 programmable DNA thermal cycler (MJ Research, Watertown MA). The amplification was performed as follows, initial 5 min 94 8C denaturation; 45 cycles of 1 min 94 8C, 1 min 52 8C, 1 min 72 8C; and 5 min 72 8C extension. Amplified fragments were separated in 1.5% agarose gel using TBE buffer and were visualized and photographed using a Gel Documentation System, GDS 8000 (BioRad, CA, USA), after staining with ethidium bromide.
2.3. Biochemical and physiological tests Isolates were characterised based on the following tests: Gram test (Sulsow et al., 1982), oxidative/fermentative test (Hugh and Leifson, 1953), production of fluorescent pigment on KB, hypersensitive reaction (HR) in tobacco and geranium leaves (Lelliot and Stead, 1987), oxidase test, levan formation, catalase, urease, gelatin liquefaction, litmus milk, salt tolerance (5%) and gas formation from glucose. In addition, tests for arginine dehydrolase, hydrogen sulfide production from peptone, reducing substance from sucrose, tyrosinase casein hydrolase, nitrate reduction, indole production, 2-keto gluconate oxidation lecitinase, starch hydrolysis, phenylalanine deaminase, esculin and Tween 80 hydrolysis and optimal growth temperature (Schaad et al., 2001). The presence of DNase was tested on DNA agar (Diagonistic Pasteur, France). On Ayer medium were also performed. Carbohydrate utilization using Ayer basal medium was carried out and the results were recorded daily up to 2–8 days (Hildebrand, 1988). For each test defined in this study, a representative isolate has been deposited in the Collection Franc¸aise de Bacte´ries Phytopathoge`ns (CFBP) culture collection. This reference isolate mainly consisted of type isolate of P. s. pv. syringae.
3. Results and discussion 3.1. Biochemical and physiological tests All isolates were Gram, oxidase, catalase negative, and unable to utilize glucose under anaerobic conditions (Table 1). None of the isolates were able to produce reducing compounds from sucrose or show lecithinase, arginine dihydrolase activity or produce gas from glucose. All isolates were aesculin positive and capable of hydrolyzing gelatin. None of the isolates were able to hydrolyze Tween 80, produce indole, reduce nitrate and oxidize 2-keto-gluconate. All isolates of P. s. pv. syringae were able to produce syringomycin and showed ice nucleation activity. All isolates were able to utilize citrate, L-Lysine and produce acid from manitol, xylose, D(+)-galactose, inositol, maltose, sorbitol, manose and sucrose. None of the isolates were capable of utilizing L-arabinose, trihalose and L-tartrate. The presence of DNase was tested on DNA agar (Diagonistic Pasteur, France). 3.2. Pathogenicity test
2.4. DNA extraction For bacterial DNA extraction, the isolates were grown overnight, in nutrient broth (Merck, Darmstadt, Germany), at 26 8C and the DNA was extracted as described by Martins et al. (2005). One tube of 1.5 ml was used to centrifuge the cells at 13,000 g for 5 min and the pellet was suspended in 200 ml Tris 0.1 mol/L and added with 200 ml of lysis solution (NaOH 0.2N and 1% SDS), mixed and deproteinized with 700 ml of phenol/ chloroform/isoamyl alcohol (25:24:1, v/v/v), homogenized and centrifuged for 10 min at 13,000 g. To precipitate DNA, 700 ml of cold isopropanol was added and spinned, washed in 70% ethanol and centrifuged. Precipitated DNA is dried at room temperature and suspended in 100 ml of water. The method described by Ausubel et al. (1996) was performed comparing 30 isolates. The samples from the both methods were electrophoresed on 1.5% agarose gels, stained with ethidium bromide and photographed under UV. 2.5. Primers for P. s. pv. syringae The 20-mer oligonucleotide PSF, 50 -TTGGCTAGGTATCGCTATGG-30 and PSR 50 -AGGACCCAGTTTTGGAGTGC-30 were designed and tested for P. s. pv. syringae (Manceau and Horvais, 1997).
After 5 months, dark sunken lesions were observed at every site inoculated with the olive isolates of P. syringae. Subsequently some of these lesions completely girdled the stems of the trees and death occurred above the inoculation point. The bacteria recovered from these lesions were identified as P. s. pv. syringae, confirming Koch’s postulates. Cankers typically ooze amber-colored gum and often become entry sites for borers (Hall et al., 2003). No ooze was observed in these lesions and no fluorescent pseudomonads were recovered. Serious infections have occurred on young trees that were wetted by rain or irrigation within a few days of planting or after suckers were removed from trunks. Frost, especially when closely followed by rain or heavy dew, leads to bacterial blast of blossoms. In areas of the world that have cool, wet winters, infection of pruning wounds during the fall or winter is common (Beers et al., 1993; Jones and Sutton, 2004). Every inoculation site developed a superficial raised growth that split the bark. It is most likely that this growth was callus tissue caused by a reaction of the olive to wounding and the presence of P. syringae. The lesions on the artificially inoculated olive plants were not observed for several months after inoculation. This long incubation period may also have occurred in the first year of infection of the olive trees in the field.
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M. Ashorpour et al. / Scientia Horticulturae 118 (2008) 128–131
Table 1 Phenotypic characteristics of Pseudomonas syringae pv. syringae isolates tested Bacterial tests Characteristics
Isolates of P. s. pv. syringae
Gram reaction Oxidative/Fermentative Fluorescent pigment HR on tobacco Ice nucleation Growth at 39 8C Syringomycin production Leaf blight on pear Pectinase Acetoin Arginine dihydrolase Levan formation Nitrate reduction Catalase Tween 80 hydrolysis Oxidase Starch hydrolysis Gelatin hydrolysis Esculin hydrolysis DNase activity Indole formation H2S from cysteine Casein hydrolysis Urease MR
+ + + + + + + + + +
Utilization of L-Lysine Citrate Lecithinase
+ +
Growth in 5% NaCl
Acid from L-Arabinose Myo-Inositol Manitol Xylose Trihalose Maltose L-Tartrate D-Galactose D-Sorbitol Sucrose D-Rafinose D-Manose D-Glucose Cellobiose Inolin Fructose Lactose Ribose D-Adnitol Glycerol
+ + + + + + + + + + +
3.3. Detection of P. s. pv. syringae by direct PCR All isolates of P. s. pv. syringae were identified by specific primers PSF and PSR. On agarose gel electrophoresis 1.5%, isolates produced a band 600 bp and all isolates of P. s. pv. syringae produced a band 600 bp (expected size). The bands of isolates were similar with isolates standards of CFBP 3077 (Fig. 1). Based on the phenotypic, pathogenicity properties and PCR tests, the causal agent of bacterial canker on olive was identified as P. s. pv. syringae. Isolates of. P. s. pv. syringae were used in this study, came from various locations within Guilan province and this is the first report of P. s. pv. syringae at high incidence on olive trees from Iran. Further biochemical and molecular work is underway to characterise the P. syringae from olives, and to test the
Fig. 1. Agarose gel electrophoresis of products from polymerase chain reaction (PCR) performed on DNA 16S of P. syringae pv. syringae isolates, M, 100 bp DNA marker; lane 1 is negative control (distilled water); lanes 2 is positive control (P. syringae pv. syringae CFBP 3077) showing the amplification the approximately 600 bp, lane 3–9, strains of P. syringae pv. syringae isolated from bacterial canker of olive.
susceptibility of other varieties. It would be difficult to study a large population of isolates in different regions of Iran. 4. Conclusion Based on morphological, phenotypical, nutritional characteristic, pathogenicity tests and PCR using specific primers, we identified causal agent of bacterial canker on olives as P. s. pv. syringae. All the isolates of P. s. pv. syringae produced canker on the stem of olive. Although the disease has occurred in several consecutive years, P. s. pv. syringae does not appear to be a widespread problem of olives in Iran. The lesions and branch death have been observed on the several varieties of olive. Further molecular work is underway to characterise the P. s. pv. syringae from olives, and to test the susceptibility of other varieties. The infection was more severe in April, with large lesions observed on the main stems and severe branch death occurred on many trees. P. s. pv. syringae was also recovered from stem lesions on 3-year-old olives cv. Marie in an adjacent planting. The lesions on the artificially inoculated olive plants were not observed for several months after inoculation. This long incubation period may also have occurred in the first year of infection of the olive trees in the orchards (Wimalajeewa and Flett, 1985). Therefore, the most likely infection period of the olives was in mid-October, where a period of high rainfall coincided with maximum temperatures of 24–28 8C. Certified olive material for export should in any case satisfy the phytosanitary regulations of importing countries, especially with respect to any of the pathogens covered by the scheme which are also quarantine pests. These bacteria were likely to have entered plants through pruning wounds or where frost/cold injury had caused stem tissue to crack or peel. PCR is considered as the most time consuming, cost effective, and rapid method for the detection and identification of pathogenic bacteria, although many improved methods (biochemical test, serological assays, fatty acids, and metabolic profiling) have been developed so far. So, the PCR assay using a primer set (PSF and PSR) designed from the sequence of hrp gene will be a useful tool for the detection and identification of P. s. pv. syringae (Manceau and Horvais, 1997). Isolates of. P. s. pv. syringae were used in this study, came from various locations within Guilan province. To our knowledge, the occurrence and incidence of this disease on olive in different geographic regions of Iran have not been studied. This is the first report of bacterial canker of olives in the Iran. It would be difficult to study a large population of isolates in different regions of Iran. Further research that elucidates the mechanisms eliciting this genetic diversity is needed. An understanding of the ecology of natural microbial communities should lead to a more efficient deployment of
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bacterial populations for disease management. Study on the biological control of bacterial blight on olive by antagonistic isolates in different parts of Iran and use of resistant cultivars could be a case study for future research. References Ausubel, F.M., Brent, R., Kingston, E., Moore, D.D., Seidmann, J.G., 1996. Current Protocols in Molecular Biology. John Wiley & Sons, New York. Beers, E.H., Brunner, J.F., Willett, M.J., Warner, G.M., 1993. Orchard Pest Management: A Resource Book for the Pacific Northwest. Good Fruit Grower, Yakima, WA. Ercolani, G.L., 1991. Distribution of epiphytic bacteria on olive leaves and the influence of leaf age and sampling time. Microbial Ecology 21, 35–48. Ferguson, L., Sibbett, G.S., Martin, G.C., 1994. Olive Production Manual. University of California, Division of Agriculture and Natural Resources: Publication 3353. Gala´n, C., Garcı´a-Mozo, H., Va´zquez, L., Ruiz, L., Dı´az de la Guardia, C., Trigo, M.M., 2005. Heat requirement for the onset of the Olea europaea L. pollen season in several sites in Andalusia and the effect of the expected future climate change. International Journal of Biometeorology 49, 184–188. Hall, B.H., Cother, E.J., Noble, D., McMahon, R., Wicks, T.J., 2003. First report of Pseudomonas syringae on olives in South Australia. Australasian Plant Pathology 32, 119–120. Hildebrand, D.C., 1988. Pectate and pectin gel for differentiation of Pseudomonas sp. and other bacterial plant pathogens. Phytopathology 61, 1430–1439.
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Hugh, R., Leifson, E., 1953. The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various Gram negative bacteria. Journal of Bacteriology 66, 24–26. Iannotta, N., Lombardo, N., Briccoli Bati, C., Monardo, D., 1999. Response of some olive cultivars to minimum late temperatures. Informatore Agrario 55, 59–62. Jones, A.L., Sutton, T.B., 2004. Bacterial canker, Pseudomonas syringae pv. syringae and P. s. pv. morsprunorum. In: Fruit Pathology, West Virginia University. Lelliot, R.A., Stead, D.E., 1987. Methods for the Diagnosis of Bacterial Disease of Plant. Blackwell Scientific Pub, London. Manceau, C., Horvais, A., 1997. Assessment of genetic diversity among strains of Pseudomonas syringae by PCR restriction fragment length polymorphism analysis of rRNA operon with special emphasis on P. syringae pv. tomato. Applied and Environmental Microbiology 63, 498–505. Martins, M.L., Arau´jo, E.F., Mantovani, H.C., Moraes, C.A., Vanetti, M.C.D., 2005. Detection of the apr gene in proteolytic psychrotrophic bacteria isolated from refrigerated raw milk. International Journal of Food Microbiology 102, 203–211. Mekuria, G.T., Collins, G.G., Sedgley, M., 1999. Genetic variability between different accessions of some common commercial olive cultivars. Journal of Horticultural Science and Biotechnology 74, 309–314. Schaad, N.W., Jones, J.B., Chun, W., 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria, Third ed. APS, St. Paul, MN, USA, p. 373. Scortichini, M., 1997. Psuedomonas syringae pv. syringae associated with decline in olive due to an excess of magnesium. Informatore Fitopatoloico 47, 47–50. Sulsow, T.V., Schorth, M.N., Saka, M., 1982. Application of a rapid method for Gram differentiation of plant pathogenic and saprophytic bacteria without staining. Phytopathology 72, 917–918. Wimalajeewa, D.L.S., Flett, J.D., 1985. A study of populations of Pseudomonas syringae pv. syringae on stone fruits in Victoria. Plant Pathology 34, 248–254.
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Bacterial canker of olives caused by of Pseudomonas syringae pv. syringae in Iran Maesomeh Ashorpour a, Mostafa Niknejad Kazempour a,*, Mohamad Ramezanie b a b
Department of Plant Pathology, Faculty of Agriculture, University of Guilan, Rasht Iran Natural Resources of Research Guilan province Rasht Iran
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 February 2008 Received in revised form 3 May 2008 Accepted 27 May 2008
Guilan is one of the main province olive oil producers in Iran. The culture of olive trees is of considerable importance. One problem that affects Iran olive orchards is a disease bacterial canker caused by Pseudomonas syringae pv. syringae. In this research, the bacterium was recovered from sunken brown stem lesions on 2-year-old olive trees cv. Marie, during survey from olive orchards in different areas of Guilan province. Samples were taken from infected tissues and were washed with sterile distilled water and crushed in peptone water. Then 50 ml of the extract was cultured on NA and King’s B medium containing cyclohexamid antibiotic (50 mg/ml). After 48–72 h, bacterial colonies were selected. Based on morphological, physiological, biochemical, pathogenicity properties and PCR methods with specific primers the predominant pathogenic type was identified as P. s. pv. syringae. This is the first report of the existence of P. s. pv. syringae on olive trees in Iran. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Bacterial canker of olive Pseudomonas syringae pv. syringae
1. Introduction Olea europaea L. is one of the oldest agricultural tree crops which is cultivated in about 71 thousand ha, situated in the north, southwest, central west and southeast regions. In recent years, molecular markers have been developed in olives. They include randomly amplified polymorphic DNA (RAPD) (Mekuria et al., 1999). The olive tree is affected by some pests and diseases, although it has fewer problems than most fruit trees. Because the olive has fewer natural enemies than other crops, and because the oil in olives retains the odor of chemical treatments, the olive is one of the least sprayed crops (Ferguson et al., 1994). Threshold temperatures varied between 5 and 12.5 8C depending on biogeographical characteristics (Gala´n et al., 2005). Bacterial stem cankers and dieback of olive caused by Pseudomonas syringae pv. syringae were likely to have entered plants through pruning wounds or where frost/cold injury had caused stem tissue to crack or peel. Symptoms vary from slow decline of trees and tree death, to localized cankers around wound sites (Hall et al., 2003). P. syringae has been reported on olives from Italy, where it has been found epiphytically on olive leaves (Ercolani, 1991) and infecting
* Corresponding author at: Department of Plant Pathology, Faculty of Agriculture, Guilan University, P.O. Box 41635-1314, Rasht, Iran. E-mail address: [email protected] (M.N. Kazempour). 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.05.035
frost damaged plants (Iannotta et al., 1999). In Southern Italy, it was found in association with necrosis and stem girdling at the collar of young trees cv. Carolea, and was attributed to a nutritional imbalance, which weakened the trees and made them susceptible to infection (Scortichini, 1997). The objectives of the present research were the isolation of the causal agent of bacterial canker on olives in the Guilan province and the identification of isolates by biochemical, nutritional, pathogenicity and PCR methods. 2. Materials and methods 2.1. Bacterial isolation Samples were collected from olive orchards in the counties Roudbar, Aliabad, Manjil and Loshan which are located in the north of Iran and have average temperature 22–25 8C and relative humidity 90–95%. Small tissue pieces from stem lesion margins, surfaces of cankers and leaf tissue showing necrotic lesions and blight symptoms were removed aseptically, ground in bacteriological saline (0.85%, w/v, NaCl), and left at room temperature (20 8C) for 10 min. Loopfuls of the bacterial suspension were streaked onto Nutrient Agar (NA) and King’s medium B (KB) and incubated at 26 8C. Pure colonies of bacteria were isolated from the lesions. Isolates were routinely grown on KB at 26 8C and stored at 4 8C for up to 2 weeks. For longer-term storage bacterial isolates were stored in freezing medium at 80 8C.
M. Ashorpour et al. / Scientia Horticulturae 118 (2008) 128–131
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2.2. Pathogenicity test on stem of olive
2.6. PCR conditions for amplification and electrophoresis
Pathogenicity was tested according to Scortichini (1997). Pathogenicity tests were conducted on young potted 18-monthold olive trees cvs. Marie, Kroniky, Conservalia and Arbkin, using 18 isolates of the olive bacterium. Stems were wounded with a 25G hypodermic needle, the wound covered with cotton wool dipped in a suspension of 1 108 bacteria/ml and the inoculation site was wrapped with grafting tape. Two isolates of P. syringae pv. syringae from cherry (DAR 30499 and 33426) were also tested for pathogenicity. A water-only inoculation was included as the control. The trees were kept in high humidity for 24 h before, and after, inoculation when the grafting tape and cotton wool were removed. The plants were then moved outdoors where they were watered overhead twice daily.
Amplification was carried out in a 25 ml volume in 0.5 ml microtube using a Hybaid programmable thermal controller. Each 25 ml PCR reaction mixture contained 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2, 200 mM of each nucleotide (dATP, dCTP, dGTP, and dTTP), 0.25 mM of each primer, 100 ng DNA and 1 U of Taq DNA polymerase (Promega Corp., Madison, WI). A 25 ml sterile, mineral-oil overlay was added to reduce evaporation. DNA amplification was carried out in a PTC-100 programmable DNA thermal cycler (MJ Research, Watertown MA). The amplification was performed as follows, initial 5 min 94 8C denaturation; 45 cycles of 1 min 94 8C, 1 min 52 8C, 1 min 72 8C; and 5 min 72 8C extension. Amplified fragments were separated in 1.5% agarose gel using TBE buffer and were visualized and photographed using a Gel Documentation System, GDS 8000 (BioRad, CA, USA), after staining with ethidium bromide.
2.3. Biochemical and physiological tests Isolates were characterised based on the following tests: Gram test (Sulsow et al., 1982), oxidative/fermentative test (Hugh and Leifson, 1953), production of fluorescent pigment on KB, hypersensitive reaction (HR) in tobacco and geranium leaves (Lelliot and Stead, 1987), oxidase test, levan formation, catalase, urease, gelatin liquefaction, litmus milk, salt tolerance (5%) and gas formation from glucose. In addition, tests for arginine dehydrolase, hydrogen sulfide production from peptone, reducing substance from sucrose, tyrosinase casein hydrolase, nitrate reduction, indole production, 2-keto gluconate oxidation lecitinase, starch hydrolysis, phenylalanine deaminase, esculin and Tween 80 hydrolysis and optimal growth temperature (Schaad et al., 2001). The presence of DNase was tested on DNA agar (Diagonistic Pasteur, France). On Ayer medium were also performed. Carbohydrate utilization using Ayer basal medium was carried out and the results were recorded daily up to 2–8 days (Hildebrand, 1988). For each test defined in this study, a representative isolate has been deposited in the Collection Franc¸aise de Bacte´ries Phytopathoge`ns (CFBP) culture collection. This reference isolate mainly consisted of type isolate of P. s. pv. syringae.
3. Results and discussion 3.1. Biochemical and physiological tests All isolates were Gram, oxidase, catalase negative, and unable to utilize glucose under anaerobic conditions (Table 1). None of the isolates were able to produce reducing compounds from sucrose or show lecithinase, arginine dihydrolase activity or produce gas from glucose. All isolates were aesculin positive and capable of hydrolyzing gelatin. None of the isolates were able to hydrolyze Tween 80, produce indole, reduce nitrate and oxidize 2-keto-gluconate. All isolates of P. s. pv. syringae were able to produce syringomycin and showed ice nucleation activity. All isolates were able to utilize citrate, L-Lysine and produce acid from manitol, xylose, D(+)-galactose, inositol, maltose, sorbitol, manose and sucrose. None of the isolates were capable of utilizing L-arabinose, trihalose and L-tartrate. The presence of DNase was tested on DNA agar (Diagonistic Pasteur, France). 3.2. Pathogenicity test
2.4. DNA extraction For bacterial DNA extraction, the isolates were grown overnight, in nutrient broth (Merck, Darmstadt, Germany), at 26 8C and the DNA was extracted as described by Martins et al. (2005). One tube of 1.5 ml was used to centrifuge the cells at 13,000 g for 5 min and the pellet was suspended in 200 ml Tris 0.1 mol/L and added with 200 ml of lysis solution (NaOH 0.2N and 1% SDS), mixed and deproteinized with 700 ml of phenol/ chloroform/isoamyl alcohol (25:24:1, v/v/v), homogenized and centrifuged for 10 min at 13,000 g. To precipitate DNA, 700 ml of cold isopropanol was added and spinned, washed in 70% ethanol and centrifuged. Precipitated DNA is dried at room temperature and suspended in 100 ml of water. The method described by Ausubel et al. (1996) was performed comparing 30 isolates. The samples from the both methods were electrophoresed on 1.5% agarose gels, stained with ethidium bromide and photographed under UV. 2.5. Primers for P. s. pv. syringae The 20-mer oligonucleotide PSF, 50 -TTGGCTAGGTATCGCTATGG-30 and PSR 50 -AGGACCCAGTTTTGGAGTGC-30 were designed and tested for P. s. pv. syringae (Manceau and Horvais, 1997).
After 5 months, dark sunken lesions were observed at every site inoculated with the olive isolates of P. syringae. Subsequently some of these lesions completely girdled the stems of the trees and death occurred above the inoculation point. The bacteria recovered from these lesions were identified as P. s. pv. syringae, confirming Koch’s postulates. Cankers typically ooze amber-colored gum and often become entry sites for borers (Hall et al., 2003). No ooze was observed in these lesions and no fluorescent pseudomonads were recovered. Serious infections have occurred on young trees that were wetted by rain or irrigation within a few days of planting or after suckers were removed from trunks. Frost, especially when closely followed by rain or heavy dew, leads to bacterial blast of blossoms. In areas of the world that have cool, wet winters, infection of pruning wounds during the fall or winter is common (Beers et al., 1993; Jones and Sutton, 2004). Every inoculation site developed a superficial raised growth that split the bark. It is most likely that this growth was callus tissue caused by a reaction of the olive to wounding and the presence of P. syringae. The lesions on the artificially inoculated olive plants were not observed for several months after inoculation. This long incubation period may also have occurred in the first year of infection of the olive trees in the field.
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M. Ashorpour et al. / Scientia Horticulturae 118 (2008) 128–131
Table 1 Phenotypic characteristics of Pseudomonas syringae pv. syringae isolates tested Bacterial tests Characteristics
Isolates of P. s. pv. syringae
Gram reaction Oxidative/Fermentative Fluorescent pigment HR on tobacco Ice nucleation Growth at 39 8C Syringomycin production Leaf blight on pear Pectinase Acetoin Arginine dihydrolase Levan formation Nitrate reduction Catalase Tween 80 hydrolysis Oxidase Starch hydrolysis Gelatin hydrolysis Esculin hydrolysis DNase activity Indole formation H2S from cysteine Casein hydrolysis Urease MR
+ + + + + + + + + +
Utilization of L-Lysine Citrate Lecithinase
+ +
Growth in 5% NaCl
Acid from L-Arabinose Myo-Inositol Manitol Xylose Trihalose Maltose L-Tartrate D-Galactose D-Sorbitol Sucrose D-Rafinose D-Manose D-Glucose Cellobiose Inolin Fructose Lactose Ribose D-Adnitol Glycerol
+ + + + + + + + + + +
3.3. Detection of P. s. pv. syringae by direct PCR All isolates of P. s. pv. syringae were identified by specific primers PSF and PSR. On agarose gel electrophoresis 1.5%, isolates produced a band 600 bp and all isolates of P. s. pv. syringae produced a band 600 bp (expected size). The bands of isolates were similar with isolates standards of CFBP 3077 (Fig. 1). Based on the phenotypic, pathogenicity properties and PCR tests, the causal agent of bacterial canker on olive was identified as P. s. pv. syringae. Isolates of. P. s. pv. syringae were used in this study, came from various locations within Guilan province and this is the first report of P. s. pv. syringae at high incidence on olive trees from Iran. Further biochemical and molecular work is underway to characterise the P. syringae from olives, and to test the
Fig. 1. Agarose gel electrophoresis of products from polymerase chain reaction (PCR) performed on DNA 16S of P. syringae pv. syringae isolates, M, 100 bp DNA marker; lane 1 is negative control (distilled water); lanes 2 is positive control (P. syringae pv. syringae CFBP 3077) showing the amplification the approximately 600 bp, lane 3–9, strains of P. syringae pv. syringae isolated from bacterial canker of olive.
susceptibility of other varieties. It would be difficult to study a large population of isolates in different regions of Iran. 4. Conclusion Based on morphological, phenotypical, nutritional characteristic, pathogenicity tests and PCR using specific primers, we identified causal agent of bacterial canker on olives as P. s. pv. syringae. All the isolates of P. s. pv. syringae produced canker on the stem of olive. Although the disease has occurred in several consecutive years, P. s. pv. syringae does not appear to be a widespread problem of olives in Iran. The lesions and branch death have been observed on the several varieties of olive. Further molecular work is underway to characterise the P. s. pv. syringae from olives, and to test the susceptibility of other varieties. The infection was more severe in April, with large lesions observed on the main stems and severe branch death occurred on many trees. P. s. pv. syringae was also recovered from stem lesions on 3-year-old olives cv. Marie in an adjacent planting. The lesions on the artificially inoculated olive plants were not observed for several months after inoculation. This long incubation period may also have occurred in the first year of infection of the olive trees in the orchards (Wimalajeewa and Flett, 1985). Therefore, the most likely infection period of the olives was in mid-October, where a period of high rainfall coincided with maximum temperatures of 24–28 8C. Certified olive material for export should in any case satisfy the phytosanitary regulations of importing countries, especially with respect to any of the pathogens covered by the scheme which are also quarantine pests. These bacteria were likely to have entered plants through pruning wounds or where frost/cold injury had caused stem tissue to crack or peel. PCR is considered as the most time consuming, cost effective, and rapid method for the detection and identification of pathogenic bacteria, although many improved methods (biochemical test, serological assays, fatty acids, and metabolic profiling) have been developed so far. So, the PCR assay using a primer set (PSF and PSR) designed from the sequence of hrp gene will be a useful tool for the detection and identification of P. s. pv. syringae (Manceau and Horvais, 1997). Isolates of. P. s. pv. syringae were used in this study, came from various locations within Guilan province. To our knowledge, the occurrence and incidence of this disease on olive in different geographic regions of Iran have not been studied. This is the first report of bacterial canker of olives in the Iran. It would be difficult to study a large population of isolates in different regions of Iran. Further research that elucidates the mechanisms eliciting this genetic diversity is needed. An understanding of the ecology of natural microbial communities should lead to a more efficient deployment of
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