First Report of Fire Blight Caused by Erwinia amylovora on Meadowsweet (Spirea prunifolia) in Turkey.

Fire blight, caused by Erwinia amylovora (Burr.) Winslow et al., affects plants in the Rosaceae family, which includes trees and shrubs in orchards, nurseries, and landscape plantations. During the springs and summers of 2008 and 2010, dying branches, necrotic leaves attached to shoots, and blighted twigs of meadowsweet (Spirea prunifolia) were observed at three different locations of landscape areas in Konya Province, Turkey. Disease incidence was approximately 1% on the plants during the surveys. Initial symptoms of reddish to brownish streaks on the shoots of infected plants were observed in spring. Nine representative bacterial strains were isolated from the lesions on shoots of seven meadowsweet plants on nutrient sucrose agar (NSA) medium and identified as E. amylovora on basis of biochemical, physiological (2,3) and molecular tests (1). Bacteria were gram-negative, rod shaped, aerobic, fermentative, yellow-orange on Miller and Scroth medium (2), positive for levan formation and acetoin production, did not grow at 36°C, positive for gelatin hydrolysis, and negative for esculin hydrolysis, indole, urease, catalase, oxidase, arginine dehydrolase, reduction of nitrate, acid production from lactose, and inositol. All strains were hypersensitive response-positive on tobacco (Nicotiana tabacum var. White Burley) plants. All strains were identified as E. amylovora using the species-specific primers set, A/B (1), by PCR assay, and by fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software (TSBA 6 v. 6.00; Microbial ID, Newark, DE) with similarity indices ranging from of 79 to 99%. Pathogenicity was tested by injecting of petioles and actively growing three shoot tips of 2-year-old S. prunifolia seedlings cv. number 29 using a 0.46 mm-diameter hypodermic needle with bacterial suspensions containing 108 CFU mL-1 in sterile distilled water (SDW) Plants were inoculated with each of the nine bacterial strains and two references strains, Ea29 and NCPPB 2791 (Selcuk University, Department of Plant Protection, Konya, Turkey). Symptoms resembling those associated with natural infection appeared on the inoculated plants 7 days after inoculation. Plants inoculated with SDW served as a negative control treatment, and no symptoms were observed on these plants. All tests were repeated three times with the same results. Bacterial re-isolations were attempted from the control plants as well as shoots and leaves inoculated with the two reference strains and the nine bacteria identified as E. amylovora. Bacteria isolated from inoculated plants were identified as E. amylovora using the biochemical, physiological, and molecular tests described above, but this bacterium was not isolated from the control plants. Phytosanitary measures must be taken to avoid spread of the pathogen to ornamentals in new landscape areas in Turkey. This report is important because infected Spirea spp. can be a potential inoculum source for other rosaceous ornamentals. To our knowledge, this is the first report of the occurrence of fire blight on meadowsweet in Turkey. References: (1) S. Bereswill et al. Appl. Environ. Microbiol. 58:3522, 1992. (2) A. L. Jones and K. Geider. Laboratory Guide for Identification of Plant Pathogenic Bacteria, pp. 40-55. American Phytopathological Society, St. Paul, MN, 2001. (3) R. A. Lelliott and D. E. Stead. Methods for Diagnosis of Bacterial Diseases of Plants (Methods in Plant Pathology). Oxford, UK, 1987.

First Report of Fire Blight Caused by Erwinia amylovora on Crabapple (Malus floribunda) in Turkey.

Fire blight is a destructive and sporadic disease of crabapple (Malus floribunda) and other plants in the Rosaceae in many areas of the world. From 2007 to 2010, sudden wilting, shriveling of flowers, leaf and shoot blight, and cankers with brown discoloration on twigs of crabapple were observed in residential landscapes of Konya Province, Turkey. Disease incidence ranged from 20 to 40% in different areas of this province, and surveys showed that ~163 ha were infested. Isolations were made from sections of symptomatic leaves, shoots, and cankers using 70% ethanol for 1 s to surface-sterilize the tissue sections, followed by rinsing three times in sterilized distilled water (SDW). Then, a 1 g subsample of each tissue section was homogenized in 10 ml phosphate buffered saline (PBS), and a 10-fold serial dilution of each homogenate prepared for six dilutions. From each homogenate, an aliquot of each dilution was plated onto 5% nutrient sucrose agar and King's B agar media, and the plates incubated for 2 to 3 days at 27°C (3). Bacterial strains were identified on the basis of biochemical, physiological (2), and molecular tests (1). Twenty-seven representative bacterial strains were each gram negative, rod-shaped, mucoid, fermentative, yellow-orange on Miller and Scroth agar medium, positive for levan formation and acetoin production, and showed no growth at 36°C. The strains were also positive for gelatin hydrolysis and negative for esculin hydrolysis, indole, urease, catalase, oxidase, arginine dihydrolase, reduction of nitrate, and acid production from lactose and inositol (2). Two reference strains of Erwinia amylovora (EaP28 and NCPPB 2791) from a culture collection at Selcuk University were used as positive control strains. All strains induced a hypersensitive response in tobacco (Nicotiana tabaccum cv. White Burley) plants within 24 h after inoculation with a 108 CFU/ml bacterial suspension in SDW (~50 µl), and the strains produced ooze on inoculated immature pear fruit slices cv. Ankara. All strains were identified as E. amylovora using the species-specific primers A/B (1), which amplified a 1 kb DNA fragment by PCR assay. Pathogenicity was confirmed by inserting a suspension (108 CFU/ml SDW) of each of the 27 bacterial strains and two reference strains, EaP28 and NCPPB 2791, into actively growing shoot tips of 3-year-old plants of M. floribunda cv. Hilleri, using a 0.46 mm-diameter hypodermic needle. Leaf and shoot blight symptoms typical of fire blight were observed within 2 weeks. SDW was injected similarly as a negative control treatment, and no symptoms were observed. All tests were repeated three times with the same results. Re-isolations were done from the control plants as well as shoots and leaves inoculated with the two reference strains and the 27 bacteria identified as E. amylovora. Bacteria isolated from inoculated plants were identified as E. amylovora using the biochemical, physiological, and molecular tests described above, but this bacterium was not isolated from the control plants. To our knowledge, this is the first report of E. amylovora on crabapple in Turkey. References: (1) S. Bereswill et al. Appl. Environ. Microbiol. 58:3522, 1992. (2) A. L. Jones and K. Geider. Laboratory Guide for Identification of Plant Pathogenic Bacteria, pp. 40-55, American Phytopathological Society, St. Paul, MN, 2001. (3) R. A. Lelliott and D. E. Stead. Methods for Diagnosis of Bacterial Diseases of Plants (Methods in Plant Pathology). Oxford, UK, 1987.

First Report of Fire Blight Caused by Erwinia amylovora on Rosehip (Rosa canina) in Turkey.

During the summers of 2008 and 2010, leaf and shoot blight, wilting of the tips of young infected shoots, and cankers with brown discoloration on twigs were observed on six dog rosehip (Rosa canina) plants from four different private orchards in Eregli district of Konya Province, Turkey. Disease incidence was estimated to be approximately 0.5% on rosehips over 2 years within all survey areas, and surveys showed that ~4 ha was infested. Bacteria isolated from diseased leaf and shoot tissues was macerated and streaked on nutrient sucrose agar (NSA) and King's medium B (KB). Typical light cream, levan-positive colonies developed on NSA medium after a 2-day incubation at 25°C. Colonies on KB were white and non-fluorescent (1). Bacterial strains were identified on the basis of biochemical, physiological (2), and molecular tests (3). Eleven representative bacterial strains isolated were gram-negative, rod-shaped, mucoid, fermentative, yellow-orange on Miller & Scroth medium, positive for levan formation and acetoin production, no growth at 36°C, positive for gelatin hydrolysis, and negative for esculin hydrolysis, indole, urease, catalase, oxidase, arginine dehydrolase, reduction of nitrate, and acid production from lactose and inositol. Two reference strains of Erwinia amylovora (Burr.) Winslow et al. (Ea43b and NCPPB 2791) obtained from culture collection of Selcuk University, Department of Plant Protection, Turkey, were used as positive controls. All strains induced a hypersensitive response in tobacco (Nicotiana tabaccum cv. White Burley) plants within 24 h after inoculation with a 108 CFU/ml bacterial suspension in sterilized distilled water (SDW) (~50 µl), and the strains produced ooze on inoculated immature pear fruit slices cv. Ankara. All strains were identified as E. amylovora using the species-specific primers set A/B (A: 5' CGGTTTTTAACGCTGGG 3' and B: 5' GGGCAAATACTCGGATT 3') (3) by PCR assay to generate a 1-kb DNA fragment, and fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software with similarity indices ranging from 84 to 97%. Pathogenicity was tested by inserting a suspension (108 CFU/ml SDW) of each of the 11 bacterial strains and two references strains into actively growing shoot tips and petioles of 4-year-old plants of Rosa canina using a 0.46-mm-diameter hypodermic needle. Leaf and shoot blight symptoms resembling the natural infection were developed on the inoculated plants 7 to 10 days after inoculation. SDW was injected similarly as a negative control treatment, and no symptoms were observed on the control plants. All tests were repeated three times. Re-isolations were done from shoots and leaves of inoculated plants with the two reference strains and the 11 bacteria, and control plants. Obtaining bacteria were identified as E. amylovora using the biochemical, physiological, and molecular tests described above, but this bacterium was not isolated from the control plants. To our knowledge, this is the first report of E. amylovora on rosehip in Turkey. References: (1) R. A. Lelliott and D. E. Stead. Methods for Diagnosis of Bacterial Diseases of Plants (Methods in Plant Pathology). Oxford, UK, 1987. (2) A. L. Jones and K. Geider. Laboratory Guide for Identification of Plant Pathogenic Bacteria, Pp. 40-55, American Phytopathological Society, St. Paul, MN, 2001. (3) S. Bereswill et al. Appl. Environ. Microbiol. 58:3522, 1992.

First Report of Bacterial Canker of Kiwifruit Caused by Pseudomonas syringae pv. actinidiae in Turkey.

A new disease was observed during the spring and autumn of 2009 and 2010 on kiwifruit plants (Actinidia deliciosa cv. Hayward) in Rize Province of Turkey. Disease incidence was estimated as 3% in approximately 10 ha. Symptoms were characterized by dark brown spots surrounded by yellow halos on leaves and cankers with reddish exudate production on twigs and stems. Eight representative bacterial strains were isolated from leaf spots and tissues under the bark on King's B medium (KB) and identified as Pseudomonas syringae pv. actinidiae on the basis of biochemical, physiological (1,2), and PCR tests (3). Bacteria were gram negative, rod shaped, and nonfluorescent on KB; positive for levan production, sucrose and inositol utilization, and tobacco (Nicotiana tabacum cv. White Burley) hypersensitivity; and negative for growth at 37°C, oxidase, potato soft rot, arginine dihydrolase, urease, arbutin, erythritol, lactic acid, aesculin hydrolysis, gelatin liquefaction, and syringomycin production. Identity of the eight isolates was confirmed by PCR using P. syringae pv. actinidiae-specific primers PsaF1/R3 to generate a 280-bp DNA fragment (3). P. syringae pv. actinidiae reference strain NCPPB 3739, and CJW7 from Jae Sung Jung, Department of Biology, Sunchon National University, Korea, were employed in all biochemical, physiological, and molecular tests as positive controls. Pathogenicity was confirmed by artificial inoculation of 2-year-old A. deliciosa cv. Hayward. A bacterial suspension (108 CFU ml-1) was injected into kiwifruit twig tips, stems, and leaves with a hypodermic syringe, and the inoculated plants were placed at 25 to 28°C and 80% relative humidity growth chamber for 3 weeks. First symptoms were observed on leaves within 5 days after inoculation and on twigs after 20 days. No symptoms were observed on control plants that were inoculated with sterile water. Reisolation was made from dark brown lesions surrounded by yellow halos on leaves and cankers on twigs and stem and their identities were confirmed using the techniques previously described. All tests were performed three times and pathogenicity tests employed three plants for each strain. To our knowledge, this is the first report of P. syringae pv. actinidiae causing disease on kiwifruit in Turkey. Kiwifruit production in Turkey has expanded rapidly during the last 10 years ( http://www.tuik.gov.tr ) and phytosanitary measures are needed to prevent further spread of the bacterium to other kiwifruit orchards. References: (1) Y. J. Koh et al. N. Z. J. Crop Hortic. Sci. 38:4, 275, 2010. (2) R. A. Lelliott and D. E. Stead. Methods for the Diagnosis of Bacterial Diseases of Plants. Blackwell Scientific, Sussex, UK, 1988. (3) J. Rees-George et al. Plant Pathol. 59:453, 2010.

First Report of Erwinia amylovora on Firethorn (Pyracantha coccinea) and Mountainash (Sorbus sp.) in Turkey.

Fire blight, caused by the bacterium Erwinia amylovora, is a serious disease of apples (Malus spp.) and pears (Pyrus spp.) but can also infect many ornamental species in the Rosaceae family. In the summers of 2009 and 2010, leaf and shoot blight and reddish colored cankers were observed on firethorn (Pyracantha coccinea) and brown discolored leaves and necrotic stem lesions on mountain ash (Sorbus sp.) both from the landscape areas of Konya province. Investigation of these symptoms showed that in an 85-ha area, disease incidence was estimated at 1.5% and 1% on firethorn and mountain ash, respectively. Bacteria were consistently isolated from both leaf and lesions onto nutrient sucrose agar medium. Nine representative bacterial colonies from firethorn isolations and six from mountain ash isolations purified and characterized as gram-negative, rod-shaped, mucoid, fermentative, yellow-orange on Miller & Scroth medium, positive for levan formation and acetoin production, no growth at 36°C, positive for gelatin and esculin hydrolysis, and negative for indole, urease, oxidase, arginine dehydrolase, reduction of nitrate, and acid production from lactose and inositol (2). Two reference strains of E. amylovora (EaP28 and NCPPB 2791) obtained from culture collection at Selcuk University, Department of Plant Protection, Konya, Turkey, were used as positive controls. All strains induced a hypersensitive response in tobacco (Nicotiana tobaccum cv. White Burley) and produced ooze when stab inoculated on immature pear fruits. In addition, all strains and the references were identified as E. amylovora on the basis of a 1-kb DNA fragment amplification with a species-specific primer set, A/B (1) in PCR. Pathogenicity tests were performed by injecting a bacterial suspension (108 CFU ml-1) into the shoot tips of 3-year-old firethorn and mountain ash seedlings, resulting in leaf and shoot blight symptoms observed 10 to 15 days after inoculation. No symptoms were observed on control plants treated with sterile water. E. amylovora was positively reisolated from leaf and shoot lesions from the inoculated seedlings and identified as described above. To our knowledge, this is the first report of E. amylovora on P. coccinea and Sorbus sp. in Turkey. References: (1) S. Bereswill et al. Appl. Environ. Microbiol. 58:3522, 1992 (2) A. L. Jones and K. Geider. Page 40 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria, 2001.

First Report of Fire Blight Disease on Blackberry in Turkey.

During 2008 and 2009, a new disease on blackberry (Rubus fruticosus cv. Chester) causing leaf and shoot blight and cankers with brown discoloration of necrotic tissues on mature branches was observed in Isparta and Konya provinces of Turkey. Disease incidence was estimated to be 4% for the two years. Isolations were made from lesions on leaves and shoots on nutrient sucrose agar (NSA) medium. Bacteria consistently isolated from the diseased tissues were identified on the basis of biochemical, physiological (2), and molecular tests (1). Eleven representative bacterial strains were gram-negative, rod-shaped, mucoid, fermentative, yellow-orange on Miller and Scroth (MS) medium, positive for levan formation and acetoin production, no growth at 36°C, positive for gelatin hydrolysis, and negative for esculin hydrolysis, indole, urease, catalase, oxidase, arginine dehydrolase, reduction of nitrate, acid production from lactose, and inositol. Two reference strains of Erwinia amylovora (EaP28 and NCPPB 2791) obtained from the culture collection unit of Selcuk University were used as positive controls. All strains induced a hypersensitive response in tobacco (Nicotiana tobaccum cv White Burley) 24 h after inoculation with a 108 CFU/ml bacterial suspension in water. All strains were identified as E. amylovora using the species-specific primers set A/B (1), which amplified a 1-kb DNA fragment in PCR, and fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software (TSBA 6 v. 6.00; Microbial ID, Newark, DE) with similarity indices ranging from of 79 to 99%. Pathogenicity was confirmed by injecting bacterial suspensions (108 CFU/ml-1) in sterile distilled water into the shoot tips of 2-year-old R. fruticosus cv. Chester and the first blighting symptoms were observed on leaves within 3 days and also 10 days later after inoculation on shoots. Sterile distilled water was used as a negative control. No symptoms were observed on control plants. All tests were repeated three times. The bacterium was reisolated from inoculated plants and identified as. E. amylovora. To our knowledge, this is the first report of E. amylovora on blackberry in Turkey. Phytosanitary measures are needed to prevent any further spread of the bacterium to new blackberry areas. References: (1) S. Bereswill et al. App. Environ. Microbiol. 58:3522, 1992. (2) A. L. Jones and K. Geider. Lab. Guide for Identification of Plant Pathological Bacteria, 40, 2001.

First Report of Fire Blight Disease Caused by Erwinia amylovora on Rockspray (Cotoneaster horizontalis) in Turkey.

In the late summer and early winter of 2008 and 2009, leaf and shoot blight and cankers with reddish and brownish necrotic tissue on mature branches of Cotoneaster horizontalis were investigated in landscape areas of Konya province in Turkey. Disease incidence was estimated at 2%. Bacteria were consistently isolated from the lesions on leaves and shoots on nutrient sucrose agar medium. Twelve representative bacterial strains were isolated and characterized as gram-negative, rod-shaped, mucoid, fermentative, yellow-orange on MS medium, positive for levan formation and acetoin production, no growth at 36°C, positive for gelatin hydrolysis, and negative for indole, urease, oxidase, arginine dehydrolase, reduction of nitrate, and acid production from lactose and inositol (2). Two reference strains of Erwinia amylovora (EaP28 and NCPPB 2791) obtained from the culture collection unit of Selcuk University were used as positive controls. All strains induced a hypersensitive response in tobacco (Nicotiana tobaccum cv. White Burley). All strains were identified as E. amylovora on the basis of amplification of a 1 kb DNA fragment with a species-specific primer set, A/B (1) by PCR, and fatty acid methyl ester profiles determined by Sherlock Microbial Identification System software (TSBA 6 v. 6.00; Microbial ID, Newark, DE) with similarity indices ranging from of 83 to 96%. Pathogenicity tests were performed by injecting 20 µl of a bacterial suspension (108 CFU ml-1) into the shoot tips of 3-year-old C. horizontalis seedlings. Leaf and shoot blighting symptoms were observed within 10 to 15 days, but no symptoms were observed on control plants treated with sterile water. The bacterium was reisolated from the lesions on leaves and shoots and identified as described above. To our knowledge, this is the first report of E. amylovora on cotoneaster in Turkey. Control measures are needed to prevent any further spread of the bacterium to new landscape areas. References: (1) S. Bereswill et al. Appl. Environ. Microbiol. 58:3522, 1992. (2) A. L. Jones and K. Geider. Page 40 in: Laboratory Guide for Identification of Plant Pathological Bacteria, 2001.

First Report of Bacterial Stalk and Head Rot Disease Caused by Pectobacterium atrosepticum on Sunflower in Turkey.

Bacterial stalk and head rot on sunflower (Helianthus annuus) was investigated in Konya Province of Turkey in 2008. Disease incidence was estimated as 30%. Bacteria appeared as droplets and ooze and symptoms were dark and water-soaked necrotic areas on stems and heads. Twenty-four strains were isolated from lesions on stalks and heads of sunflower cv. TR3080 from a 25-ha field and identified as Pectobacterium atrosepticum (formerly Erwinia caratovora subsp. atroseptica) (2) on the basis of biochemical, physiological (3), and molecular tests (1). Bacteria were gram negative, rod shaped, fermentative, nonfluorescent on King's B medium; positive for gelatin liquefaction, CVP test, catalase, and pectolytic activity, growth on 5% NaCl, reducing substances from sucrose, acid-production from lactose and α-methyl glucoside; and negative for growth at 37°C, acid production from sorbitol and maltose, phosphatase activity, tests for egg yolk (lecithin), sensitivity to erythromycin, and pigmentation on yeast dextrose calcium carbonate agar medium. To distinguish between P. atrosepticum and P. carotovorum, particular attention was paid to the growth at 37°C, reducing substances from sucrose and the utilization of α-methyl glucoside. Mesophyll cells of tobacco plants (Nicotiana tobaccum cv. White Burley) were infiltrated with bacterial suspensions (108 cells/ml) or water (control). Brown, collapsed areas of tissues (hypersensitive response) were observed at the injection sites after incubation for 48 h at 28°C and 80% relative humidity. A P. atrosepticum-specific primer set, Y45/Y46 (3), was used in PCR reactions to generate a 439-bp DNA fragment. Reference strains, Eca17 from Aegean University, Department of Plant Protection (Izmir, Turkey) and NCPPB 1277 from Selcuk University, Department of Plant Protection, Konya, Turkey, were employed in all biochemical, physiological, and molecular tests as positive controls and similar results were obtained. Koch's postulates were carried out to establish a causal relationship between the bacteria and the disease. A bacterial suspension (108 CFU/ml) was injected into sunflower shoot tips and inoculated plants were incubated for 2 weeks at 28°C and 80% relative humidity. All bacterial strains obtained from the stalks and heads produced the rot symptoms and ooze following inoculation to the susceptible sunflower cv. TR 3080. No symptoms were observed on controls that were inoculated with sterile water. The bacteria were isolated from the lesions on stalks and heads and their identities confirmed by the biochemical, physiological, and molecular tests. All tests were performed three times on three plants per strain. To our knowledge, this is the first report of P. atrosepticum on sunflower in Turkey. Further research is needed to determine how far the disease is spread in Turkey since other provinces also grow sunflowers. References: (1) L. Gardan et al. Int. J. Syst. Evol. Microbiol. 53:381, 2003. (2) L. Hauben et al. Syst. Appl. Microbiol. 21:384, 1998. (3) A. Darrasse et al. Appl. Environ. Microbiol. 60:298, 1994.