K. K. Bastas

A Genome-Wide Functional Investigation into the Roles of Receptor-Like Proteins in Arabidopsis

Receptor-like proteins (RLPs) are cell surface receptors that typically consist of an extracellular leucine-rich repeat domain, a transmembrane domain, and a short cytoplasmatic tail. In several plant species, RLPs have been found to play a role in disease resistance, such as the tomato (Solanum lycopersicum) Cf and Ve proteins and the apple (Malus domestica) HcrVf2 protein that mediate resistance against the fungal pathogens Cladosporium fulvum, Verticillium spp., and Venturia inaequalis, respectively. In addition, RLPs play a role in plant development; Arabidopsis (Arabidopsis thaliana) TOO MANY MOUTHS (TMM) regulates stomatal distribution, while Arabidopsis CLAVATA2 (CLV2) and its functional maize (Zea mays) ortholog FASCINATED EAR2 regulate meristem maintenance. In total, 57 RLP genes have been identified in the Arabidopsis genome and a genome-wide collection of T-DNA insertion lines was assembled. This collection was functionally analyzed with respect to plant growth and development and sensitivity to various stress responses, including susceptibility toward pathogens. A number of novel developmental phenotypes were revealed for our CLV2 and TMM insertion mutants. In addition, one AtRLP gene was found to mediate abscisic acid sensitivity and another AtRLP gene was found to influence nonhost resistance toward Pseudomonas syringae pv phaseolicola. This genome-wide collection of Arabidopsis RLP gene T-DNA insertion mutants provides a tool for future investigations into the biological roles of RLPs.

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 Kings 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.

Bacterial leaf and peduncle soft rot caused by Pectobacterium carotovorum on tulips in Konya, Turkey

The etiology of a new soft rot disease of tulips, causing leaf spots, leaf blight, neck and bulb rot in Konya, Turkey, was investigated. This disease occurred in tulip fields grown for seed bulbs of various varieties in 2002. Bacteria were isolated from the lesions on leaf, bud neck and bulbs and the causal bacterium was identified asPectobacterium carotovorum on the basis of biochemical and physiological tests. Cells were Gram-negative, rod-shaped, fermentative, potato-rot positive. Colonies were capable of growth at 37°C. On nutrient agar the colonies were creamy-white. The isolates were non-fluorescent on King’s B medium, positive for acetoin production, gelatin liquefaction, acid-production-from-lactose, and catalase; and negative for gas from glucose, reducing substances from sucrose, and phosphatase activity. Additionally, tests for egg yolk (lecithin), sensitivity to erythromycin, and pigmentation on yeast dextrose carbonate agar were negative; growth on 5% sodium chloride was positive. All the bacterial isolates obtained from the leaf, bud neck and bulbs produced the original symptoms following inoculation to the susceptible tulip variety ‘Gander’. The rate of damage caused by this bacterium was evaluated on several tulip varieties under field and storage conditions. Gander was the most susceptible variety in the field whereas ‘Salmon Parrot’ exhibited the highest rate of bulb rot in storage. Disease severity was lower in 2003 than 2002.

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 Kings 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 (İzmir, 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. Kochs 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.

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 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.