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

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 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 Rosehip (Rosa canina) in Turkey

Plant Disease ◽

10.1094/pdis-03-13-0273-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1652-1652 ◽

Cited By ~ 2

Author(s):

K. K. Bastas ◽

F. Sahin ◽

R. Atasagun

Keyword(s):

Erwinia Amylovora ◽

Hypodermic Needle ◽

Bacterial Suspension ◽

Identification System ◽

Bacterial Strains ◽

Rosa Canina ◽

First Report ◽

Molecular Tests ◽

Shoot Blight ◽

Reference Strains

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 Isolation of Exiguobacterium aurantiacum in Serbia

Polish Journal of Microbiology ◽

10.33073/pjm-2021-037 ◽

2021 ◽

Vol 70 (3) ◽

pp. 405-407

Author(s):

VERA P. GUSMAN ◽

DEANA D. MEDIĆ ◽

ANIKA DJ TRUDIĆ ◽

PAVLE Z. BANOVIĆ ◽

NATAŠA M. NIKOLIĆ

Keyword(s):

Mass Spectrometry ◽

Environmental Samples ◽

Automated System ◽

Distilled Water ◽

Immunocompromised Patients ◽

Bacterial Strains ◽

Ionization Time ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Rare Bacteria

Exiguobacterium aurantiacum is isolated from a variety of environmental samples but rarely from patients. The aim of the study was to represent isolation of unusual bacterial strains that could cause infection in patients. Final identification was performed using matrix-assisted description/ionization time-of-flight mass spectrometry (MALDI-TOF). Two isolates strains of E. aurantiacum were isolated, one isolate from distilled water used during surgical treatment and the second one from a patient with bacteremia after radical prostatectomy, both sensitive to all tested antimicrobials. Environmental strains could cause infection, especially in immunocompromised patients; therefore, rare bacteria testing is required, in which identification special assistance is provided by an automated system MALDI-TOF.

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An Improved Method for Rapid Detection of Mycobacterium abscessus Complex Based on Species-Specific Lipid Fingerprint by Routine MALDI-TOF

Frontiers in Chemistry ◽

10.3389/fchem.2021.715890 ◽

2021 ◽

Vol 9 ◽

Author(s):

Min Jia Khor ◽

Agnieszka Broda ◽

Markus Kostrzewa ◽

Francis Drobniewski ◽

Gerald Larrouy-Maumus

Keyword(s):

Ribosomal Proteins ◽

Mycobacterium Abscessus ◽

Bacterial Suspension ◽

Rapid Diagnostics ◽

Maldi Tof ◽

Appropriate Treatment ◽

Subspecies Level ◽

Successful Recovery ◽

The Matrix ◽

Species Specific

Rapid diagnostics of bacterial infection is the key to successful recovery and eradication of the disease. Currently, identification of bacteria is based on the detection of highly abundant proteins, mainly ribosomal proteins, by routine MALDI-TOF mass spectrometry. However, relying solely on proteins is limited in subspecies typing for some pathogens. This is the case for, for example, the mycobacteria belonging to the Mycobacterium abscessus (MABS) complex, which is classified into three subspecies, namely, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Being able to detect bacteria accurately and rapidly at the subspecies level could not only reliably identify the pathogen causing the disease but also enable better antibiotic stewardship. For instance, M. abscessus subsp. abscessus and M. abscessus subsp. bolletii possess a functional erm41 (erythromycin ribosomal methylation gene 41) gene, whilst M. abscessus subsp. massiliense does not, resulting in differences in macrolide antibiotic (e.g., clarithromycin and azithromycin) susceptibilities. This presents a challenge for physicians when designing an appropriate treatment regimen. To address this challenge, in addition to proteins, species-specific lipids have now been considered as a game changer in clinical microbiology diagnostics. However, their extraction can be time-consuming, and analysis requires the use of apolar toxic organic solvents (e.g., chloroform). Here, we present a new method to accurately detect species and subspecies, allowing the discrimination of the mycobacteria within the MABS complex and relying on the use of ethanol. We found that a combination of the matrix named super-DHB with 25% ethanol with a bacterial suspension at McFarland 20 gave robust and reproducible data, allowing the discrimination of the bacteria within the MABS complex strains tested in this study (n = 9). Further investigations have to be conducted to validate the method on a larger panel of strains for its use in diagnostic laboratories.

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First Report of Shoot Blight and Canker Caused by Diplodia coryli in Hazelnut Trees in Chile

Plant Disease ◽

10.1094/pdis-07-12-0667-pdn ◽

2013 ◽

Vol 97 (1) ◽

pp. 144-144 ◽

Cited By ~ 3

Author(s):

J. A. Guerrero ◽

S. M. Pérez

Keyword(s):

Sodium Hypochlorite ◽

Control Plant ◽

Width Ratio ◽

Pathogenicity Test ◽

Distilled Water ◽

Dark Light ◽

First Report ◽

Agar Plug ◽

Rdna Sequencing ◽

Shoot Blight

Shoot blight, twig and branch cankers with grayish discoloration, roughened tissue, and dieback were observed in 5-year-old European hazelnut trees (Corylus avellana L.) cv. Barcelona at two commercial plantations in the Allipén locality, Region de La Araucanía (38° 59′ 24.76″ S, 72° 29′ 35.58″ W), Chile, during the 2011 to 2012 growing season. In addition, discoloration of the wood was observed in cross-sections. In order to isolate the causal agent, entire twigs and shoot fragments with cankers were sterilized in 0.5% sodium hypochlorite for 2 minutes, followed by two rinses with sterile distilled water. The tissues were then stored in a humid chamber. Sub-epidermal black pycnidia with sporulation were detected on the symptomatic tissue. Conidia were transferred to potato dextrose agar (PDA) (Difco, Lawrence, KS) and incubated at 25°C in the dark. The mycelia were black, creeping, and compact in appearance. The mature conidia were dark brown with a single septum, slightly constricted at the septum, and ovoid with a broadly rounded apex; some had a truncated base. Conidia had the following measurements: (20.0-) 23.1 ± 1.9 (-28.0) × (10-) 11.9 ± 1.2 (-15) μm with an average length/width ratio of 1.95 ± 0.17 (n = 50). These morphometric characteristics correspond to those of Diplodia coryli Fuckel (1870), teleomorph: Botryosphaeria sensu lato. The identity of the fungus was confirmed using internal transcribed spacer (ITS) rDNA sequencing completed at CABI, United Kingdom. The sequencing report indicated that the isolate (IMI-501235a) had 100% homology with a reference strain (CBS 242.51) in the CBS collection. The obtained sequence was deposited in GenBank (Accession No. JX163116). The anamorphs of Botryosphaeria have been divided into up to 18 genera (1), many of which are not clearly defined. Diplodia (3), including D. coryli (CBS 242.51) and D. juglandis (CBS 188.87), have been included within the genus Dothiorella (2), but the taxon names have not been formally changed. A pathogenicity test was conducted with one isolate (IMI-501235a) and four 1-year-old plants of hazelnut cultivar Barcelona. Plants were maintained in individual bags in greenhouse conditions (14/10 hours dark/light, 20°C; 70% RH). Prior to inoculation, plant tissues were externally disinfected with sodium hypochlorite (2%) and rinsed with sterile distilled water. Each plant was inoculated at fresh wound sites on two shoots and three twigs around each vegetative bud. The inoculum consisted of an agar plug with mycelia (5 mm) from the margin of an actively growing colony cultured on PDA media for 7 days. Each wound was covered with moistened cotton and sealed with Parafilm; a control plant was inoculated in the same way with agar only. After 3 months, fragments of necrotic and discolored vascular system tissues from inoculated shoots were removed and incubated on PDA. D. coryli was consistently recovered from these tissues, satisfying Koch's postulates. The control plant showed no symptoms of the disease. D. coryli has been reported to cause symptoms of dieback (dead branches) in Italy and Spain. To our knowledge, this is the first report of D. coryli on C. avellana cv. Barcelona in Chile. European hazelnut is an emerging crop in Chile, grown mainly for export, and management strategies for this disease will need to be developed. References: (1) S. Denman et al. Stud. Mycol. 45:129, 2000. (2) A. J. L. Phillips et al. Persoonia 21:29, 2008. (3) A. J. L. Phillips et al. Mycologia 97:513, 2005.

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First Report of Colletotrichum acutatum Associated with Stem Blight of Blueberry Plants in China

Plant Disease ◽

10.1094/pdis-08-12-0738-pdn ◽

2013 ◽

Vol 97 (3) ◽

pp. 422-422 ◽

Cited By ~ 3

Author(s):

C.-N. Xu ◽

Z.-S. Zhou ◽

Y.-X. Wu ◽

F.-M. Chi ◽

Z.-R. Ji ◽

...

Keyword(s):

Vaccinium Corymbosum ◽

Colletotrichum Acutatum ◽

Post Inoculation ◽

Pathogenicity Test ◽

Distilled Water ◽

Conidial Suspension ◽

First Report ◽

Rdna Its ◽

Its Sequence Analysis ◽

Species Specific

An anthracnose disease was observed on stems of high-bush blueberry plants (Vaccinium corymbosum L.) in Liaoning Province, China in 2012. The typical symptoms consist of sudden wilting and dieback of stems during the growing season. Dark brown lesions originate from infected buds and kill portions of the stems. Lesions have grayish white centers, with the necrotic areas becoming 6 to 8 cm in length. Disinfected stem pieces were placed on potato dextrose agar (PDA) and incubated at 28°C for 5 to 7 days, after which the emerging colonies were transferred to fresh PDA. All isolates initially produced white growth, but turned pink after 7 days before becoming blackish green. The average colony diameter was 65.5 to 75.0 mm after 7 days. Conidia were aseptate, hyaline, fusiform to ellipsoid, 8.5 to 16.5 × 2.5 to 4.0 μm in size and single celled with two to seven oil globules. Setae were not found on the acervuli. These characteristics matched published descriptions of Colletotrichum acutatum (1) (teleomorph Glomerella acutata). Pathogenicity test was confirmed in 15 2-year-old healthy potted plants of cv. Berkeley. Stems of 10 plants were punctured with flamed needles and sprayed with 5 ml of conidial suspension (106 conidia per ml in sterile distilled water) of isolate LNSW1. Five control plants were inoculated with sterile distilled water. Seven days after inoculation, eight of the 10 blueberry plants exhibited stem lesions, leaf chlorosis, followed by branch dieback 15 days post-inoculation. The symptoms were similar to those observed on diseased plants in the field, and no lesions were observed on control plants. The pathogen was reisolated from the margin of lesions and identified by colony growth characteristics on PDA. PCR amplification of one isolate (LNSW1) was carried out by utilizing the universal rDNA-ITS primer pair ITS1/ITS4. The sequence (557 bp) of isolate LNSW1 (GenBank Accession No. JX392857) showed 99% identity to G. acutata (AB443950) and C. acutatum (AJ749672) in a BLAST search. An approximately 490-bp fragment was amplified from LNSW1 by the species-specific primer pair CaInt2/ITS4 (2). The pathogen was identified as G. acutata (asexual stage: C. acutatum J.H. Simmonds) on the basis of morphological characters, rDNA-ITS sequence analysis, and a PCR product with species-specific primers. To our knowledge, this is the first report of C. acutatum in high-bush blueberry plants in China. References: (1) C. Lei et al. Fungal Diversity 12:183, 2009. (2) S. Sreenivasaprasad et al. Plant Pathol. 45:650, 1996

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First Report of Pseudomonas syringae pv. coriandricola Causing Bacterial Leaf Spot on Carrot, Parsley, and Parsnip in Serbia

Plant Disease ◽

10.1094/pdis-10-14-1041-pdn ◽

2015 ◽

Vol 99 (3) ◽

pp. 416-416 ◽

Cited By ~ 6

Author(s):

T. Popović ◽

Ž. Ivanović ◽

M. Ignjatov ◽

D. Milošević

Keyword(s):

Pseudomonas Syringae ◽

Leaf Spot ◽

Bacterial Suspension ◽

Pathogenicity Test ◽

Distilled Water ◽

Bacterial Leaf Spot ◽

Koch’S Postulates ◽

First Report ◽

Vegetable Farm ◽

Koch's Postulates

During the spring of 2014, a severe leaf spot disease was observed on carrot (Daucus carota), parsley (Petroselinum crispum), and parsnip (Pastinaca sativa) on a 0.5-ha vegetable farm in Vojvodina Province, Serbia. The disease appeared under wet and cool conditions with 5 to 25% of plants infected for each of the three crops. Symptoms were characterized as brown angular leaf spots, ~2 mm in diameter, often limited by veins. Collected symptomatic leaves were rinsed and dried at room temperature, and leaf sections taken from the margin of necrotic tissue were macerated in sterile phosphate buffer and streaked onto nutrient agar with 5% (w/v) sucrose (NAS). After isolation, whitish, circular, dome-shaped, Levan-positive colonies consistently formed. Five strains from each host (carrot, parsley, and parsnip) were used for further study. Strains were gram-negative, aerobic, and positive for catalase and tobacco hypersensitive reaction but negative for oxidase, rot of potato slices, and arginine dihydrolase. These reactions corresponded to LOPAT group Ia, which includes Pseudomonas syringae pathovars (3). Repetitive extragenic palindromic sequence (Rep)-PCR fingerprint profiles using the REP, ERIC, and BOX primers (4) were identical for all strains. Sequence typing of the housekeeping genes gyrB and rpoD (1) was performed for three representative strains (one from each host). Sequences were deposited in the NCBI GenBank database as accessions KM979434 to KM979436 (strains from carrot, parsnip, and parsley, respectively) for the gyrB gene and KM979437 to KM979439 (strains from parsnip, parsley and carrot, respectively) for the rpoD gene. Sequences were compared with pathotype strain Pseudomonas syringae pv. coriandricola ICMP12471 deposited in the Plant Associated and Environmental Microbes Database ( http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl ). BLAST analysis revealed 100% homology for gyrB and 99% homology for rpoD. Pathogenicity was tested with five representative strains from each host on four-week-old plants of carrot (cv. Nantes), parsley (cv. NS Molski), and parsnip (cv. Dugi beli glatki) using two methods: spraying the bacterial suspension (108 CFU ml−1) on the leaves until runoff (5) and injecting the bacterial suspension into leaves with a hypodermic syringe (2). Four plants were used per strain and method. Sterile distilled water was applied as a negative control treatment for each plant species. All plants were kept in a mist room with 100% humidity for 4 h, then transferred to a greenhouse at 25°C and 80% relative humidity and examined for symptom development over a period of three weeks. For all strains, inoculated leaves first developed water-soaked lesions on the leaves 5 to 7 days after inoculation (DAI); 14 DAI lesions became dark brown, often surrounded by haloes. No symptoms were observed on control plants inoculated with sterile distilled water. For fulfillment of Koch's postulates, re-isolations were done onto NAS. Re-isolated bacteria were obtained from each inoculated host and confirmed to be identical to the original isolates using the LOPAT tests and Rep-PCR fingerprinting profiles. Based on the pathogenicity test accompanied by completion of Koch's postulates, sequence analysis, and bacteriological tests, the strains were identified as P. s. pv. coriandricola. To our knowledge, this is the first report of bacterial leaf spot of carrot, parsley, and parsnip in Serbia. It may present a threat to production due to quality requirements for fresh market. References: (1) P. Ferrente and M. Scortichini. Plant Pathol. 59:954, 2010. (2) M. Gupta et al. Plant Dis. 97:418, 2013. (3) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (4) F. J. Louws et al. Appl. Environ. Microb. 60:2286, 1994. (5) X. Xu and S. A. Miller. Plant Dis. 97:988, 2013.

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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 Xanthomonas campestris pv. campestris as the causal agent of necrotic leaf spot in Phaseolus vulgaris at Puebla, Mexico

Plant Disease ◽

10.1094/pdis-05-21-1010-pdn ◽

2021 ◽

Author(s):

Conrado Parraguirre-Lezama ◽

Omar Romero Arenas ◽

Maria de los Angeles Valencia de Ita ◽

Antonio Rivera ◽

Nemesio Villa-Ruano ◽

...

Keyword(s):

Leaf Spot ◽

Xanthomonas Campestris ◽

Casamino Acid ◽

Bacterial Suspension ◽

Pathogenicity Test ◽

Distilled Water ◽

Tetrazolium Chloride ◽

First Report ◽

Bean Plants ◽

Xanthomonas Campestris Pv Campestris

Beans are the most cultivated legume in the world. In Mexico, it is the second most important crop after corn (FAO 2020; SIAP 2020). Bean plants “Flor de Mayo M38” variety were affected by a foliar disease during the agricultural cycle 2019 in Puebla-Mexico (19°02'46.6" LN and 98°05'15.6" LO). Necrotic V- shaped lesions were observed on the margins of the leaves surrounded by yellow halos followed by foliar necrosis, affecting 40% of the crop. In Mexico this variety of cultivars is in great demand for local consumption and generates income in foreign currency (Castellanos et al. 1997). Sampling was carried out on 50 plants “Flor de Mayo M38” variety, with necrotic leaf symptoms from ten plots of one hectare. Samples were cut into pieces (5 mm), disinfested with 1% hypochlorite 3 min, and washed with sterile distilled water. Subsequently, samples were dried on sterile paper and placed on Petri plates containing yeast extract calcium carbonate dextrose agar (YDC) medium and kept at 36°C for 3 days. Colonies of ten typical bacteria isolated from all symptomatic plants were Gram (-), small and uniform in size with rounded edges, yellow, convex with entire borders and mucoid appearance on YDC. Bacteria did not grow on 0.1% triphenyl tetrazolium chloride amended casamino acid, peptone, and glucose medium (CPG). Biochemical tests showed that isolates did not reduce nitrate to nitrites, had positive catalase and starch hydrolysis, while the Kovac oxidase test was negative (Schaad and White 1974). Genus identity of the representative isolate Xcf1-APJR, was confirmed by 16S rRNA encoding gene partial sequencing, using universal primers 518F (5'-CCAGCAGCCGCGGTAATACG-3') and 800R (5′-TACCAGGGTATCTAATCC-3′) (Halim et al. 2020). BLASTn alignments against the nucleotide collection were 100% identical to Xanthomonas sequences including Xanthomonas campestris pv. campestris strains NZ_AP019684.1, CP025750.1, and MN108237.1. The 1,418 bp sequence was deposited in the GenBank database under accession number MT645246. The identification of species/pathovar was accomplished by serological methods using a polyclonal antiserum specific for X. campestris pv. campestris (Popovic ́ et al. 2013) with the DAS-ELISA commercial kit (catalog number 07122C/096, LOEWE Biochemica GmbH, Germany). The pathogenicity test was carried out on 50 healthy bean plants from the "Flor de Mayo M38" variety. Bacterial culture incubated at 28°C for 48 h in YDC medium was used to prepare the bacterial suspension (108 CFU mL-1). The first two lower leaves of 30-day-old plants were inoculated by sprinkling. Ten plants sprayed with sterile distilled water were used as negative control. All plants were kept for 20 days in greenhouse at 18-26°C and relative humidity of 60%. After seven days, chlorotic lesions developed on all inoculated plants that became necrotic from 14 days after inoculation (dai). Necrotic leaf spots merged at 14 dai to form necrotic areas of more than 20 mm in diameter, reaching total necrosis of the leaf tissue at 20 dai and were similar to the symptoms observed in the field. Koch's postulates were confirmed by the reisolation of Xcf1-APJR strain, which presented the same colony morphology, partial sequence, and polyclonal specific detection. This is the first report of this pathogen causing necrotic leaf spot in beans from the "Flor de Mayo M38" variety in Puebla-Mexico. The author(s) declare no conflict of interest. References: FAO. 2020. FAOSTAT. Food and Agriculture Data. http://www.fao.org/faostat/en/#home/. SIAP. 2020. Atlas Agroalimentario. https://www.gob.mx/siap/. Castellanos, J. Z., et al. 1997. Arch. Latinoam. Nutr. 47:163. Schaad, N. W., and White, W. C. 1974. Phytopathology. 64:876. https://doi.org/10.1094/Phyto-64-876 Halim, R. A., et al. 2020. HAYATI J. Biosciences. 27:215. https://doi.org/10.4308/hjb.27.3.215 Popovic ́, T., et al. 2013. Plant Dis. 97:418. https://doi.org/10.1094/PDIS-05-12-0506-PDN

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