ISSN 1313 - 8820Volume 7, Number 1

March 2015

2015

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Editors and Sections

Genetics and Breeding

Atanas Atanasov (Bulgaria)Nikolay Tsenov (Bulgaria)Max Rothschild (USA)Ihsan Soysal (Turkey)Horia Grosu (Romania)Bojin Bojinov (Bulgaria)Stoicho Metodiev (Bulgaria)

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Nikolai Todorov (Bulgaria)Peter Surai (UK)Zervas Georgios (Greece)Ivan Varlyakov (Bulgaria)

Production Systems

Dimitar Pavlov (Bulgaria)Bogdan Szostak (Poland)Dimitar Panaiotov (Bulgaria)Banko Banev (Bulgaria)Georgy Zhelyazkov (Bulgaria)

Agriculture and Environment

Georgi Petkov (Bulgaria)Ramesh Kanwar (USA)Martin Banov (Bulgaria)

Product Quality and Safety

Marin Kabakchiev (Bulgaria)Stefan Denev (Bulgaria)Vasil Atanasov (Bulgaria)

English Editor

Yanka Ivanova (Bulgaria)

2015

ISSN 1313 - 8820 Volume 7, Number 1March 2015

Occurrence of Pseudomonas syringae pv. tomato in Bulgaria

1 1 2 1M. Stoyanova *, K. Aleksandrova , D. Ganeva , N. Bogatzevska

1Institute of Soil Science, Agrotechnologies and Plant Protection, Nikola Pushkarov, 7 Shosse Bankya, 1331 Sofia, Bulgaria2Vegetable Crops Research Institute Maritsa, 32 Brezovsko shose, 4000 Plovdiv, Bulgaria

Abstract. Bacterial speck of tomato caused by Pseudomonas syringae pv. tomato is an important disease in many tomato growing areas. The pathogen is known to comprise two races – R0 and R1. Sixty-seven pathogenic strains isolated in the period 2000 – 2013 from tomatoes with symptoms of bacterial speck in Bulgaria were the object of this study. The isolates were identified as P. syringae pv. tomato by the miniaturized Biolog system and possess the typical metabolic profile for the species. The pathogenic population of P. syringae pv. tomato in Bulgaria is homogeneous by phenotypic characteristics. The races of the strains were determined. On the base of the obtained results and the data available in the literature for the country, the proportions of the occurrence of the two races since 1985 alter cyclically with a tendency for shortening the cycle. The changes in the spread and development of the disease are related to the climatic changes.

Keywords: Pseudomonas syringae pv. tomato, occurrence, races dynamics

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 7, No 1, pp , 2015141 - 144

Introduction Materials and methods

Bacterial speck of tomato caused by Pseudomonas syringae Plant material and isolation procedurepv. tomato is an important disease in many tomato growing areas Plant samples were collected from growing and selection fields which is favored by cool, moist environmental conditions. in the regions of Sadovo (Vegetable Crops Research Institute Characteristic symptoms of the bacterial speck are necrotic spots Maritsa), Pazardzik, Haskovo, Sofia (Institute of Plant Physiology surrounded by chlorotic halos on leaves, as well as scabby lesions and Genetics), Kostinbrod, Pavlikeni, Byala Cherkva, Petrich and on infected fruits. The disease ultimately results in yield loss, mostly Blagoevgrad yearly from 2000 – 2013. Bacteria were isolated from due to reduced photosynthetic capacity of infested foliage and from leaves, petioles, stems, flowers and fruits of tomatoes with lesions on the fruit that render them unsuitable for the fresh market symptoms of bacterial speck with serial dilutions on King's B medium (Louwis et al., 2001; Wilson et al., 2002; Bogatzevska, 2002; as described by Rudolph et al. (1990). Pure cultures were stored at Milijasevic et al., 2009). 4ºC on potato-dextrose agar (PDA).

The pathogen retains and disseminates with tomato seeds and weeds. P. syringae pv. tomato develops symptomless epiphytically Pathogenicity testson the leaf surface and ednophytically in leaf tissues of tomato host The pathogenic potential of the bacterial strains was examined plants and in non-host weed associations. The disease does not by infiltration of tobacco cv. Samsun NN (Klement, 1963) and emerge each year but the bacteria still survives in resident phase artificial inoculation of tomato plants cv. Milyana by bacterial

4with the roots of perennial weeds (Bogatzevska, 2002). Two races suspensions of 10 cfu/ml from 36 h cultures grown on PDA. Tomato R0 and R1 of P. syringae pv. tomato have been determined. The plants in phase 5–6 – leaf were infiltrated by the vacuum infiltration pathogen races were reported in Canada (Lawton and Mac Neill, method (Bogatzevska et al., 1989) in five repeats per bacterial 1986), Italy (Buonario et al., 1996), California, USA (Arredondo and isolate and placed in Knop's solution (Kiray et al., 1974) in laboratory Daves, 2000), Turkey (Sahin, 2001), Tanzania (Shenge et al., 2007), conditions at room temperature (20 – 25°С). Hypersensitive Serbia (Milijasevic et al., 2009), Nepal (Lamichhane et al., 2009) and reaction of tobacco leaves was observed on 18, 24 and 36 h. Portugal (Cruz et al., 2010). Both races were established on the Symptoms of bacterial speck of leaves, petioles and stems were

thterritory of Bulgaria (Bogatzevska et al., 1989, 2000; Bogatzevska, observed on the 4 – 5 day after the inoculation.2002).

Data on the features of the pathogen development cycle and Biochemical characteristics and identificationthe different sensitivity of tomato cultivars to P. syringae pv. tomato The pathogenic isolates were identified by the differentiation races is necessary for selection of resistant varieties and lines. At the scheme of Schaad et al. (2001) for Pseudomonas syringae pvs. same time poor information is available worldwide on pathogen Identification of the isolates was confirmed by the miniaturized

TMcharacteristics population heterogeneity, race composition and its identification system Biolog (Biolog™, USA) with GN2 emergence in time. MicroPlate™ test plates. The v4.20.05 of the software program

This study aims to present a thorough metabolic pattern of MicroLog™ (Biolog™, USA) was used. P. syringae pv. tomato Bulgarian P. syringae pv. tomato population and to reveal the International Collection of Microorganisms from Plants 2844 was occurrence of the two races in Bulgaria in the past decade and the used as reference. The data from metabolic profiles was further dynamics of the race structure throughout the years. cluster analyzed through the SPSS procedure by the Ward's

141

* e-mail: mimka@gbg.bg

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method. The matrix of similarity between the isolates was calculated raffinose, pyruvic аcid methyl еster, D-glucuronic acid, β-using the Squared Euclidean distance. hydroxybutiric acid, α-ketobutyric acid, malonic acid, glucuronamid,

acetic acid, L-histidine, L-leucine, L-ornithine, trehalose, D-serine, Races L-threonine and α-glycerol phosphate.Races of P. syringae pv. tomato were determined by the method The reaction of the strains to ten substrates shows some

of Bogatzevska et al. (1989). Differentiator tomato lines Chico relevance to the race belonging (Table 1, Figure 1). The population (sensitive) and Ontario 7710 (resistant to R0) were used (Lawton of P. syringae pv. tomato is homogeneous as the strains shared 2/3 and Mac Neill, 1986). Plants in phase 3 –5 leaf were infiltrated by the of their metabolic patterns and reacted variably to only 17% of all vacuum infiltration method (Bogatzevska et al., 1989) with bacterial tested substrates.

4suspensions of 10 cfu/ml from 36 h cultures grown on PDA. The The race composition of the pathogen in Bulgaria includes both th the two races which occur differently through the years (Figure 2). reaction was observed on the 4 – 5 day after the inoculation. Watery

Previous investigations reveal predominance of R0 in the period chlorotic lesions with necrotic centers were considered as sensitivity 1985 – 1990 and in the next 9-year period the disease developed (S) and lack of reaction was considered as resistance (R). only in one year – 1995. In the newly investigated period starting from 2000 the first occurrence of bacterial speck was recorded in 2005 on tomato seedlings. In the next years the disease was Results and discussionobserved in small number of plants in growing and selection fields in combination with pathogenic xanthomonads on young leaves and An overall of 67 isolated bacterial strains induced

th green fruits of mature plants. In this period bacterial speck was hypersensitive reaction on tobacco leaves on the 18 hour and overcome by mass occurrence of bacterial spot (Kizheva et al., typical for bacterial speck lesions on leaves, petioles and stems of 2011). On the basis of the data in the literature and the new results tomato test plants. The strains were Gram-negative, oxidase-the disease characterizes with severe outbreaks in the 1970s – negative aerobs, synthesizing a fluorescent pigment on King's B 1980s (Vitanov, 1974; Bogatzevska 2002) and occurs significantly medium, lacking arginine dihydrolase and pectolytic activity on less in the period from 1990 until now. The occurrence of disease potato slices. D-alanine, trehalose and L-valine were not used as lacked also in the years 2007 and 2010 – 2012. The high sole carbon sources and D-sorbitol, sucrose and glucose were temperatures and relatively dry weather do not favor the metabolized which are typical characteristics of P. syringae. development of pathogenic phase of the pathogen which is an Additional differential tests to determine the pathovar were explanation of disease appearance fluctuations. The relevance of carried out. The isolated strains are ice-nucleation negative, the disease symptoms, climatic changes and cultivar sensitivity was degrade sodium polipectate at pH 5 and do not assimilate erythritol previously established by Boneva (1991). and L-tartrate. The isolates can use D-tartrate and D-mannitol as

The occurrence of R0 and R1 in pathogenic phase throughout sole carbon sources. According to their properties, the strains were the years in Bulgaria has wavy dynamics which seems to be identified as P. syringae pv. tomato.independent from blank periods in disease occurrence (Figure 2). For confirmation purposes and biochemical characterization 35 The proportions of the incidence of the two races from 1985 until strains were blindly selected for further analyses. All of them were 2013 alter cyclically with equal distribution of both races followed by identified with Biolog™ system as P. syringae pv. tomato with a predominance of one of the races, then the next equal distribution probability 82–100%, similarity index 0.506 – 0.932 and distance of both races followed by a predominance of the other race. The index 0.31 – 7.18. The reference strain showed probability 100%, dynamics keep their alteration, in the years the disease is present similarity index 0.932 and distance index 1.01. although the maximum amplitude of the proportions differs and there Biolog metabolic characteristics of the strains have been is a tendency for shortening the cycle. The predominance of R0 had obtained. Common characteristics of all the strains were utilization its maximum in 1985 – 1987 with more than 98% evidence of L-arabinose, D-arabitol, D-fructose, D-galactose, α-D-glucose, (Bogatzevska, 2002) and in 2008 R0 had 75% evidence. Kunkeaw m-inositol, D-mannitol, D-mannose, sucrose, D-sorbitol, cis-aconitic

acid, citric acid, D-gluconic acid, quinic acid, D-saccharic acid, alaninamide, D-аlanine, L-alanine, L-alanyl- glycine, L-asparagine, L-aspartic acid, L-glutamic аcid, L-proline, pyruvic acid, L-serine, γ-aminobutyric acid, inosine, uridine, and α-keto glutaric acid. Reaction to Tween 40 and Tween 80 was weak but still positive. The isolated strains did not use as a sole carbon source α-cyclodextrin, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, cellobiose, i-еrythritol, L-fucose, gentibiose, α-D-lactose, lactulose, maltose, D-melibiose, β-methyl-D-glucoside, turanose, xylitol, L-rhamnose, lactic acid, α-hydroxybutiric acid, p-hydroxyphenylacetic acid, sebacic acid, glycyl-L-aspartic acid, hydroxy-L-proline, phenylalanine, carnitine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, D-glucose-1-phosphate and D-glucose-6-phosphate.

The strains weakly or strongly utilized bromosuccinic acid, urocanic acid, glycerol, glycil-L-glutamic аcid, and succinic аcid methyl еster. The strains did not react or reacted weakly to glycogen, α-ketovaleric acid, itaconic acid. Differences in assimilation (positive, weak positive or negative) were observed to psicose, D-

Table 1. Differences in assimilation of substrates included TMin Biolog according to the race of P. syringae pv. tomato

+ positive; V+ positive or weak positive; V- negative or weak positive; - negative

Substrate R0 R1

dextrin

trehalose

aconitic acid

formic acid

D-galactonic acid lactone

D-glucosaminic acid

γ-hydroxybutiric acid

propionic acid

succinic acid

succinamic acid

-

V-

V-

V+

V+

V+

V+

+

V+

V+

V-

-

V+

+

+

+

+

V+

+

+

Figure 1. Cluster analysis of isolated P. syringae pv. tomato strains according to their race and the differences in assimilation of substrates

59g

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et al. (2010) report an increasing problem in California due to the R0 in this period – in 2008, R1 still caused 25% of disease cases evolution and prevalence of highly aggressive R1 strains from the compared to only 1.4% in 1985 – 1987. On the bases of these data end of XX century until now. Although we registered a prevalence of and climatic changes with more dry days and higher temperatures

100%

90%

80%

70%

60%

50%

40%

30%

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10%

0%

1985

-198

7

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-199

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race R0

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Figure 2. Dynamics in occurrence of R0 and R1 of P. syringae pv. tomato in Bulgaria

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throughout the year it is possible that the next period with R0 peak Kiray Z, Klement Z, Shoymoshi F and Veresh I, 1974. Methods of evidence has even lower amplitude than in 2008 and the incidence phytopathology, Kolos, Moskow.of R1 is higher. There are no resistant to R1 varieties in the world. If Kizheva Y, Vancheva T, Hristova P, Stoyanova M, Bogatzevska R1 proves to be more aggressive than R0 in the next decades, the

NS and Moncheva P, 2011. Diversity of Xanthomonas spp causal selection of resistant varieties will be placed in a challenge.

agents of bacterial spot on pepper and tomato plants in Bulgaria.

Biotechnology and Biotechnological Equipment, 25, 98-104.Klement Z, 1963. Rapid detection of the pathogenicity of

Conclusion phytopathogenic Pseudomonads. Nature, 199, 299-300.Kunkeaw S, Tan S and Coaker G, 2010. Molecular and

The pathogenic population of P. syringae pv. tomato in Bulgaria evolutionary analyses of Pseudomonas syringae pv. tomato race 1. is homogeneous by phenotypic characteristics and heterogeneous Molecular Plant Microbe Interactions, 23, 415-424.by race composition. The proportions of the occurrence of the two Lamichhane JR, Kshetri MB, Mazzaglia A, Varvaro L and races from 1985 until 2013 alter cyclically, independently from blank Balestra GM, 2009. Bacterial speck caused by Pseudomonas periods in disease occurrence, with a tendency for shortening the syringae pv. tomato race 0: first report in Nepal. New Disease cycle and increased incidence of R1 in the periods with R0 Reports, 19, 34. prevalence. Blank periods in disease occurrence are related to the Lawton MB and Mac Neill BH, 1986.Occurrence of Race 1 of changes in climatic conditions. Pseudomonas syringae pv. tomato on field tomato in southwestern

Ontario. Canadian Journal of Plant Pathology, 8, 85-88.Louws F, Wilson M, Campbell H, Cuppels D, Jones J,

References Shoemaker P, Sahin E and Miller S, 2001. Field control of bacterial spot and bacterial speck of tomato using a plant activator. Plant

Arredondo C and Daves S, 2000. First report of race 1 on tomato in Disease, 85,481-488.California. Plant Disease, 84, 371-372. Milijasevic S, Todorovic B, Rekanovic E, Potocnik I and Bogatzevska NS, Griesbach E and Sotirova V, 2000. Race of the Gavrilovic V, 2009. Races and hosts of Pseudomonas syringae pv. natural pathogenic population of Pseudomonas syringae pv. tomato tomato in Serbia. Archives of Biological Sciences, 61, 93-103.and resistance to tomato lines in Bulgaria. Reports of Bulgarian Rudolph K, Roy MA, Sasser M, Stead DE, Davis M, Swings J and Academy of Sciences, 53, 85-88. Gossele F, 1990. Chapter 1.4. Isolation of bacteria. In: Methods in Bogatzevska NS, 2002. Plant pathogenic bacteria from genus Phytobacteriology. (eds. Z. Кlement, K. Rudolph, and D. Sands). Pseudomonas group syringae and genus Xanthomonas group Akademiai Kiado, Budapest.vesicatoria and axonopodis-phases of the life cycle. Thesis for DSc, Sahin F, 2001. Severe outbreak of bacterial speck, caused by Institute of Soil Science, Agrotechnologies and Plant Protection N. Pseudomonas syringae pv. tomato, on field-grown tomatoes in Pushkarov, Sofia (Bg). eastern Anatolia region of Turkey. New Disease Reports, 3, 9. Bogatzevska NS, Sotirova V and Stamova LD, 1989. Race of Schaad NW, Jones J and Chun W, 2001. Laboratory Guide for Pseudomonas syringae pv. tomato (Okabe) Young et al. Reports of Identification of Plant Pathogenic Bacteria, Third edition. APS Press, Bulgarian Academy of Sciences, 42, 129-130. St. Paul, Minnesota, USA.Boneva P, 1991. Bacterial speck of tomato Pseudomonas syringae Shenge KC, Stephan D, Mabagala RB, Mortensen CN and Wydra pv. tomato (Okabe) Young et al- epidemiology and control in the K, 2008. Molecular Characterization of Pseudomonas syringae pv. region of Veliko Tarnovo. Thesis for PhD, Sofia (Bg). tomato Isolates from Tanzania. Phytoparasitica, 36, 338-351.Buonaurio R, Stravato V and Cappelli C, 1996. Occurrence of Vitanov M, 1974. Bacterial speck on tomato. Plant protection, 22, Pseudomonas syringae pv. tomato tomato race 1 in Italy on Pto gene 40-41 (Bg).bearing tomato plants. Journal of Phytopathology, 144, 437-440. Wilson M, Campbell P, Jones J and Cuppels D, 2002. Biological Cruz L, Cruz J, Eloy M, Oliveira H, Vaz H and Tenreiro R, 2010. control of bacterial speck of tomato under field conditions at several First Report of Bacterial Speck of Tomato Caused by Pseudomonas locations in North America Phytopathology, 92, 1284-1292.syringae pv. tomato Race 1 in Portugal. Plant Disease, 94, 1504.

Review

Genetics and Breeding

Nutrition and Physiology

Production Systems

Molecular mechanisms and new strategies to fight stresses in egg-producing birds E. Shatskikh, E. Latypova, V. Fisinin, S. Denev, P. Surai

Gene action in the inheritance of date to ear emergence and time to physiological maturity in bread wheat crosses (Triticum aestivum L.)N. Tsenov, T. Gubatov, E. Tsenova

Productivity and stability of the yield from common winter wheat cultivars developed at IPGR Sadovo under the conditions of Dobrudzha region P. Chamurliyski, E. Penchev, N. Tsenov

Effectof black (stem) rust (Puccinia Graminis F.SP. Tritici) attack to the spike characteristics in Polishwheat (Triticum Polonicum L.)H. Stoyanov

Analysis of DNA polymorphism of CAST gene in Local Karnobat and Stara Zagora sheep breedsD. Hristova, S. Georgieva, S. Tanchev

Correlation between grain yield and yield components in winter barley varieties N. Markova Ruzdik, D.Valcheva, D.Vulchev, Lj. Mihajlov, I. Karov, V. Ilieva

Genetic diversity in different accessions of oat (Avena sativa L.)T. Savova, B. Dyulgerova, G. Panayotova

Interspecific hybridization in cotton and its use in breedingA. Stoilova, I. Saldzhiev

Influence of the direction of crossing on heterosis and transgression events in relation to the length of the vegetative period of Burley tobaccos variety group Y. Dyulgerski, T. Radoukova, L. Dospatliev

The performance of female dairy calves fed texturized starters with different protein sources E. Yavuz, N. Todorov, G. Ganchev, K. Nedelkov

Feeding value estimation of spring forage pea (Pisum sativum L.) in organic cultivationI. Nikolova, N. Georgieva, Y. Naydenova

Treatment of post harvest residues with cellulose decomposing preparationsI. Effect on grain yield from wheatG. Milev, I. Iliev, A. Ivanova

CONTENTS 1 / 2

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Seasonal dynamics of important for Coriandrum sativum virus pathogensB. Dikova, H. Lambev

Crop relationship "yield-evapotranspiration" for green beanR. Kalaydzhieva, D. Davidov, A. Matev, V. Kuneva

Species composition and density of weeds in a wheat crop depending on the soil tillage system in crop rotationP. Yankov, M. Nankova, M. Drumeva, D. Plamenov, B. Klochkov

Assessment of Bulgarian Black Sea coastal water using the biological quality element phytoplanktonD. Petrova, D. Gerdzhikov

Evaluation on reaction of late maturing maize hybrids and lines to Fusarium ear rotM. Haddadi, M. Zamani

Contemporary state of macrophytobenthos along the Bulgarian coast of the Black SeaD. Petrova, V. Vachkova, D. Gerdzhikov

Bioconversion of nitrogen in eco-technical system for eggs productionA. Gencheva

Mixed viral infections in tomato as a precondition for economic lossN. Petrov

Storage and its effect on the antioxidant capacity of dried Bulgarian Chrysanthemum balsamita L.A. Popova, D. Mihaylova, I. Alexieva

Correcting the breadmaking quality of flour damaged by Sunn pest (Eurygaster integriceps) by using apple pectinI. Stoeva

Investigation the influence of dietary fiber on the rheological properties of alginate beadsZ. Manev, N. Petkova, P. Denev, D. Ludneva, S. Zhelyazkov

Occurrence of Pseudomonas syringae pv. tomato in BulgariaM. Stoyanova, K. Aleksandrova, D. Ganeva, N. Bogatzevska

Agriculture and Environment

Product Quality and Safety

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thmance in dairy cows,IX International Conference on Production Diseases in Farm Animals, September 11–14, Berlin, Germany.Thesis:Hristova D, 2013. Investigation on genetic diversity in local sheep breeds using DNA markers. Thesis for PhD, Trakia University, Stara Zagora, Bulgaria, (Bg).

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