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.

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

Plant Disease ◽

10.1094/pdis-08-12-0790-pdn ◽

2013 ◽

Vol 97 (9) ◽

pp. 1244-1244

Author(s):

K. K. Bastas ◽

A. Y. Ozturk

Keyword(s):

Fire Blight ◽

Erwinia Amylovora ◽

Hypodermic Needle ◽

Bacterial Suspension ◽

Bacterial Strains ◽

First Report ◽

Molecular Tests ◽

Malus Floribunda ◽

Shoot Blight ◽

Reference Strains

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.

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

Plant Disease ◽

10.1094/pdis-03-13-0220-pdn ◽

2014 ◽

Vol 98 (1) ◽

pp. 153-153 ◽

Cited By ~ 2

Author(s):

K. K. Bastas ◽

F. Sahin

Keyword(s):

Pathogenic Bacteria ◽

Fire Blight ◽

Erwinia Amylovora ◽

Natural Infection ◽

Hypodermic Needle ◽

Identification System ◽

Bacterial Strains ◽

First Report ◽

Molecular Tests ◽

Initial Symptoms

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.

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

Plant Disease ◽

10.1094/pdis-01-12-0002-pdn ◽

2012 ◽

Vol 96 (11) ◽

pp. 1690-1690

Author(s):

K. K. Bastas ◽

F. Sahin

Keyword(s):

Erwinia Amylovora ◽

Disease Incidence ◽

Acid Methyl Ester ◽

Late Summer ◽

Control Measures ◽

Bacterial Suspension ◽

Identification System ◽

Bacterial Strains ◽

First Report ◽

Microbial Identification

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.

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Konya İli Böğürtlen, Ahududu ve Kuşburnu Yetiştiriciliğinde Potansiyel Bir Tehdit: Ateş Yanıklığı Hastalığı

Turkish Journal of Agriculture - Food Science and Technology ◽

10.24925/turjaf.v9isp.2663-2669.4932 ◽

2022 ◽

Vol 9 (sp) ◽

pp. 2663-2669

Author(s):

Aysun Öztürk ◽

Kubilay Kurtulus Bastas

Keyword(s):

Bacterial Suspension ◽

Pathogenicity Test ◽

Distilled Water ◽

Bacterial Strains ◽

Molecular Tests ◽

Flight Mass Spectrometry ◽

The Matrix ◽

Shoot Blight ◽

Phytosanitary Measures ◽

Species Specific

In the present study, totally 49 samples, which showed the symptoms of leaf and shoot blight and cankers with brown discoloration of necrotic tissues on mature branches, were collected from 22 districts and areas of Konya Province between 2017 and 2019. Presence rate of E. amylovora in collected samples, showing symptoms of the disease, from the province was determined to be 40% for blackberry and raspberry and 33% rosehip for rosehip in three years. Bacteria consistently isolated from the diseased tissues were identified on the basis of biochemical, physiological, and molecular tests, comparing with a reference strain of E. amylovora, isolated from blackberry (Kbb 371). Twenty seven representative bacterial strains were gram-negative, rod-shaped, mucoid, fermentative, 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. All strains induced a hypersensitive response in tobacco (Nicotiana tobacum cv. White Burley) 24 h after inoculation with a 108 CFU ml-1 bacterial suspension in sterile distilled water. The 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 the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method. In order to fulfill the Koch postulates, pathogenicity test was confirmed by injecting bacterial suspensions of 108 CFU ml-1 in sterile distilled water into the shoot tips of 3-year-old blackberry R. fruticosus cv. Chester, raspberry R. idaeus cv. Heritage and rosehip R. canina. All tests were repeated three times. The bacterium was re-isolated from inoculated plants and identified as E. amylovora. Phytosanitary measures are needed to prevent any further spread of the bacterium as potential inoculum sources to new blackberry, raspberry and rosehip growing areas.

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

Plant Disease ◽

10.1094/pdis-08-11-0675 ◽

2012 ◽

Vol 96 (3) ◽

pp. 452-452 ◽

Cited By ~ 37

Author(s):

K. K. Bastas ◽

A. Karakaya

Keyword(s):

Pseudomonas Syringae ◽

Disease Incidence ◽

Soft Rot ◽

Bacterial Suspension ◽

Bacterial Strains ◽

First Report ◽

Molecular Tests ◽

Leaf Spots ◽

National University ◽

Phytosanitary Measures

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.

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First Report of Fire Blight Disease on Blackberry in Turkey

Plant Disease ◽

10.1094/pdis-12-11-1073-pdn ◽

2012 ◽

Vol 96 (12) ◽

pp. 1818-1818

Author(s):

K. K. Bastas ◽

F. Sahin

Keyword(s):

Disease Incidence ◽

Acid Methyl Ester ◽

Negative Control ◽

Culture Collection ◽

Bacterial Suspension ◽

Identification System ◽

Distilled Water ◽

Bacterial Strains ◽

First Report ◽

Microbial Identification

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.

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

Plant Disease ◽

10.1094/pdis-01-12-0014-pdn ◽

2012 ◽

Vol 96 (12) ◽

pp. 1818-1818

Author(s):

K. K. Bastas

Keyword(s):

Pathogenic Bacteria ◽

Erwinia Amylovora ◽

Plant Protection ◽

Disease Incidence ◽

Culture Collection ◽

Bacterial Suspension ◽

First Report ◽

Mountain Ash ◽

Shoot Blight ◽

Pyracantha Coccinea

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.

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First Report of Fire Blight on Pear Trees Caused by Erwinia amylovora in Kurdistan Province, Iran

Plant Disease ◽

10.1094/pdis-09-12-0876-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1111-1111 ◽

Cited By ~ 1

Author(s):

S. N. Mollaei ◽

B. Harighi

Keyword(s):

Fire Blight ◽

Erwinia Amylovora ◽

Reca Gene ◽

Phylogenetic Position ◽

Bacterial Suspension ◽

Sodium Hypochlorite Solution ◽

Pathogenicity Test ◽

First Report ◽

Appl Environ ◽

Pear Trees

Pear (Pyrus L.) is one of the most widely grown crops in western Iran. Since 2010, an outbreak of a disease with symptoms similar to fire blight has been observed on pear trees in various locations of Kurdistan Province. Initial flower symptoms include water-soaking and rapidly shriveling, infected flowers that remained hanging on the trees. Immature fruits become water-soaked, turned brown, and shriveled. Infected flowers and immature fruits were collected from different locations in the province. Small pieces (about 1 mm2) were excised from infected tissues, surface sterilized with 0.5% sodium hypochlorite solution, followed by rinsing in sterile-distilled water (SDW). Each piece was macerated in 2 to 3 ml of SDW, streaked onto nutrient agar sucrose or eosin methylene blue agar media, and incubated at 27 to 29°C. After 48 to 72 h, single colonies were subcultured onto the same media and stored at 4°C. In total, 74 bacteria were isolated from infected tissues. All isolates were gram-negative and rod-shaped. Based on other phenotypic properties, strains were grouped into three clusters at a similarity level of 65% (data not shown). Forty-one and 23 strains showed properties as expected for Erwinia amylovora and Enterobacter sp., respectively. Other strains showed properties resembling Pantoea agglomerans. All strains identified as E. amylovora produced an expected DNA fragment of about 900 bp by PCR using primers PE29A and PE29B corresponding to plasmid pEA29 (1). The result was confirmed by using primers AMSbL and AMSbR derived from the ams region required for amylovoran synthesis of E. amylovora. E. amylovora strains produced an expected 1,600-bp fragment (2). For the pathogenicity test, a bacterial suspension was adjusted to approximately 1 × 107 CFU/ml from cell cultures grown in nutrient broth at 27°C for 48 h. Immature pear fruits sterilized with 70% ethanol and rinsed with SDW were injected with the bacterial suspension using a 25-gauge sterile needle. Fruits injected with sterile water were used as controls. Pear fruits were kept in a mist chamber at 27 to 29°C. Symptoms were assessed up to 2 weeks after inoculation. All E. amylovora strains produced typical symptoms on inoculated immature pear fruits. Necrosis and oozing of bacterial exudates were observed after 3 to 7 days. The phylogenetic position of two selected strains was analyzed by sequence comparison of recA gene among other species in the genus Erwinia and related bacteria. The recA sequence of bacterial strains identified as E. amylovora revealed high similarity (99%) to the E. amylovora type strain (CFBP 1430). Genetic diversity of selected strains was assessed and compared with E. amylovora reference strain CFBP 1430 using ERIC and REP primers in rep-PCR analysis. (3). UPGMA cluster analysis of the combined data obtained in the rep-PCR experiments using Dice's coefficient revealed that the majority of E. amylovora strains showed the same fingerprint patterns at a similarity level of 93%, indicating genetic homogeneity among strains but clearly separated from Enterobacter sp. and P. agglomerans strains. To our knowledge, this is the first report that characterizes the phenotypic and genetic properties of E. amylovora in western part of Iran. References: (1) S. Bereswill et al. Appl. Environ. Microbiol. 58:3522, 1992. (2) S. Bereswill et al. Appl. Environ. Microbiol. 61:2636, 1995. (3) J. Versalovic et al. Mol. Cell Biol. 5:25, 1994.

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First Report of Bacterial Fruit Blotch of Watermelon in Oregon

Plant Disease ◽

10.1094/pdis.1997.81.1.113a ◽

1997 ◽

Vol 81 (1) ◽

pp. 113-113 ◽

Cited By ~ 9

Author(s):

P. B. Hamm ◽

D. S. Spink ◽

G. H. Clough ◽

K. S. Mohan

Keyword(s):

Citrullus Lanatus ◽

Nutrient Agar ◽

Green Water ◽

Hypodermic Needle ◽

Identification System ◽

Columbia Basin ◽

Plastic Mulch ◽

First Report ◽

Bacterial Fruit Blotch ◽

Test Plants

Most of the watermelons, Citrullus lanatus (Thunb.) Matsum. & Nakai, consumed in the Pacific Northwest during the summer months are grown in the southern Columbia Basin under dry (<5 cm rainfall), low relative humidity (46 to 57%), and high temperature (29 to 41°C) conditions, using transplants, plastic mulch, and drip irrigation. During May 1996, irregularly shaped, water-soaked lesions were observed on cotyledons and first true leaves of watermelon cv. Sangria transplants growing in a greenhouse. Similar lesions were observed later on older leaves in a commercial field of cv. Millionaire. Microscopic examination of symptomatic tissue revealed bacterial streaming, and isolation on nutrient agar consistently yielded numerous creamy to off-white bacterial colonies. Bacteria from purified, single colonies were Gram negative and rod shaped. Physiological characterization by the Biolog GN Bacterial Identification System (version 3.5) showed a similarity of 0.971 to the Biolog description for Acidovorax avenae subsp. citrulli. Pathogenicity of two strains was confirmed in three separate tests by hypodermic needle infiltration of cotyledons or by stab inoculation into hypocotyls of 12 to 24 21-day-old cv. Crimson Sweet seedlings with aqueous suspensions of bacteria containing approximately 6.0 × 108 CFU/ml. Inoculum was prepared from 48-h-old nutrient agar cultures. Test plants were incubated in the greenhouse at 21°C, under a 16-h photoperiod. Hypocotyl and cotyledon inoculations produced water-soaked lesions within 24 to 48 h on both the hypocotyl and cotyledons or just the cotyledon, respectively, on plants inoculated by either method. No symptoms developed on control plants infiltrated or stabbed with sterile water only. Isolations from three symptomatic seedlings yielded colonies similar in morphology to those used for inoculation. Tests of two purified cultures by Biolog indicated the bacteria were A. avenae subsp. citrulli. The symptomatic test plants were transplanted to fields, and the maturing melons developed large, dark green, water-soaked lesions with irregular margins. Similar fruit symptoms were seen in commercial fields. Labels on seed used in commercial production and in our tests warned of risks related to fruit blotch. This is the first report of bacterial fruit blotch of watermelon in Oregon. This disease may have a significant impact on watermelon production in the Columbia Basin.

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First Report of Crown Gall, Caused by Agrobacterium tumefaciens on Soapberry (Sapindus delavayi) in China

Plant Disease ◽

10.1094/pdis-10-12-0988-pdn ◽

2013 ◽

Vol 97 (5) ◽

pp. 685-685

Author(s):

Y. J. Wang ◽

Y. Y. He ◽

Z. Xie ◽

L. Q. Zhang

Keyword(s):

Agrobacterium Tumefaciens ◽

16S Rdna ◽

Crown Gall ◽

Maximum Parsimony Analysis ◽

Bacterial Suspension ◽

Identification System ◽

Distilled Water ◽

Bacterial Identification ◽

Poor Growth ◽

First Report

Soapberry (Sapindus delavayi (Franch.) Radlk.,) plants are widely grown as shade trees in the subtropical to tropical regions of China. In July 2011, large, aerial galls were observed on the above-ground trunks of 5-year-old soapberry plants in two commercial nursery gardens located in Zhejiang Province. Disease incidence was estimated to be 75%. The galls varied in weight from 2 to 24 g and in texture from soft and spongy to hard, and in some cases, the galls completely girdled the trunk. The trees with galls exhibited poor growth compared with healthy trees. Isolations from the grinded and macerated galls yielded nearly pure white, circular, and glistening bacterial colonies on Roy Sauer medium (2). Six random colonies from different galls were selected for bacterial identification, and showed the same morphological, physiological, and biochemical characters and 16S rDNA sequences. All six isolates (isolate SD01 to SD06) were gram negative, rod-shaped bacteria. Carbon source utilization testing with the Biolog GN Bacterial Identification System (version 3.50) confirmed the bacteria as Agrobacterium tumefaciens with a similarity of 0.90. The most-parsimonious tree from the maximum parsimony analysis (PHYLIP package, version 3.68, 500 replicates) of bacterial 16S rDNA gene sequences showed that A. tumefaciens SD01 (GenBank Accession No. JX997939) clustered phylogenetically most closely (99.5% similarity) with A. tumefaciens C58 (AE007870.2). Pathogenicity was confirmed by injecting 3- to 5-week old tomato and sunflower plants and 2-year-old soapberry with approximately 5 μl of the bacterial suspension (108 CFU/ml) in sterile, distilled water. Sterile distilled water was used as a negative control. Ten plants of each treatment were inoculated. Inoculated plants were then transferred to a greenhouse at 25°C. Typical tumors developed at the inoculation sites on tomatoes and sunflower plants 3 weeks after inoculation and on soapberry 6 weeks after inoculation. No symptoms were observed on the control plants. The bacteria that were readily reisolated from the inoculated plants exhibited the same morphological, physiological characters and 16S rDNA sequence as the original culture and were confirmed as A. tumefaciens, fulfilling Koch's postulates. A. tumefaciens is endemic to China and has a very wide host range (1). However, crown gall of soapberry has never been found in China and other countries. To our knowledge, this is the first report of A. tumefaciens on soapberry plants in China. References: (1) M. A. Escobar and A. M. Dandekar. Trends Plant Sci. 8:380, 2003. (2) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001.

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