Pathotypes Research Articles

Genetic diversity of Plasmodiophora brassicae pathotype based on newly developed SSR markers

Genetic diversity of Plasmodiophora brassicae pathotype based on newly developed SSR markers

Genomic Features and Lytic Activity of the Bacteriophage PPPL-1 Effective against Pseudomonas syringae pv. actinidiae, a Cause of Bacterial Canker in Kiwifruit.

Bacterial canker in kiwifruit is caused by Pseudomonas syringae pv. actinidiae (Psa). In this study, the bacteriophage PPPL-1 effective against Psa was characterized. Belonging to the Podoviridae family, PPPL-1 was effective against most Psa strains as well as most Pseudomonas syringae pathovars. PPPL-1 carries a 41,149-bp genome with 49 protein coding sequences and is homologous to the previously reported phiPSA2 bacteriophage. The lytic activity of PPPL-1 was stable up to 40°C, within a range of pH 3-11 and under 365 nm UV light. These results indicate that the bacteriophage PPPL-1 might be useful to control Psa in the kiwifruit field.

Analysis of the molecular diversity of R. solani isolates from five Ugandan agro-ecological zones using inter - simple sequence repeat markers

This study used seven inter-simple sequence repeat (ISSR) markers to characterize 55 R. solani isolates in order to establish their genetic diversity. The isolates were from five common bean growing agro-ecological zones of Uganda; the Southwest highlands (SW), Lake Victoria crescent and Mbale farmlands (LVC), Teso farming system (TFS), the Northern mixed farmlands (N) and the Western highlands and Semliki flats (W). DNA was extracted from 7-day old R. solani isolates growing on potato dextrose agar and amplified using PCR. Molecular analysis based on the ISSR fingerprints established moderate genetic diversity (mean = 0.389 ± 0.022). In addition, a high degree of intraspecific variation was observed by cluster analysis which generated a dendrogram with 5 highly reliable clusters (p ≥ 0.95) whose composition was independent of agro-ecological zones. Further evidence of intraspecific variation was provided by analysis of molecular variance which ascribed all the observed variation to genetic differences within the isolates. Isolates from SW, W, TFS and N had significant standardized index of association values indicative of clonal reproduction while isolates from LVC did not have significant values whence were sexual. The genetic diversity revealed by our study means that screening common bean germplasm for sources of resistance to R. solani should be done using diverse pathotypes and secondly, the breeders should pyramid many disease resistance genes in elite cultivars to provide durable resistance against R. solani.

REP-PCR DISTINGUISHES RICE BACTERIAL BLIGHT PATHOGEN (XANTHOMONAS ORYZAE pv. ORYZAE) STRAINS OF INDIAN MAINLAND AND BAY ISLANDS

Thirty-two strains of Xanthomonas oryzae pv. oryzae (Xoo) representing 12 pathotypes from major rice growing Indian mainland states and Andaman Islands were analyzed using RepPCR and IS1112-based PCR approaches. All four PCR primers (BOX, REP, ERIC and JEL primers) confirmed the presence of high genetic variability among the Xoo strains studied. The REP primers generated more PCR fragments than other set of primers and the amplification pattern of BOX, REP, ERIC and JEL primers were 277, 171, 147 and 149 respectively with the band size varying from 200 to 3000 base pairs. Cluster analysis of 32 Xoo strains revealed that the combined dendrogram constructed using Rep–PCR analysis was effective in distinguishing Islands Xoo strains from Indian mainland strains with the similarity index of Islands clusters of about 53%. In case of IS1112 repeat element based PCR approach, the two of the mainland strains (DX_027 and DX_143) were grouped with one mainland isolate ANBB_4 in one sub cluster whereas remaining three mainland isolates grouped separately in a main cluster. This indicated that Islands strains were genetically distinct from mainland strains. Also, the Rep-PCR approach was found to be more precise and thus suitable for discriminating the variability among Xoo strains emanating from geographically diverse locations.

IDENTIFICATION AND PATHOGENICITY OF PSEUDOMONAS SYRINGAE GENOMOSPECIES 1 PHYLOGROUP 2B CAUSING LEAF SPOTS AND FRUIT WARTS ON CUCURBITS IN WESTERN WASHINGTON, U.S.

Pseudomonas syringae pv. lachrymans and pv. syringae have been identified as pathogenic on cucurbit crops in the United States and based on molecular data, pv. syringae (genomospecies 1) are genetically distinct from pv. lachrymans (genomospecies 2). In 2015, leaf lesions, and bull’s-eye lesions and raised warts on fruit were observed on ‘Cinnamon Girl’ pumpkins near Mount Vernon in western WA. PsAs_2015 was collected from leaf lesions and PsFt1_2015 and PsFt2_2015 were collected from fruit. The isolates were phenotypically and molecularly characterized. LOPAT results were consistent for P. syringae. Neighbor joining and Bayesian analyses of gltA and gyrB sequences clustered the three isolates within genomospecies 1 phylogroup 2b. ‘Cinnamon Girl’ seedlings inoculated with these isolates were spotted with small lesions having chlorotic borders. Seedlings and fruits of 11 pumpkin and squash cultivars were inoculated with PsAs_2015. Altogether, 100% disease incidence occurred on five cultivars 2 (Baby Bear, Baby Boo, Cinnamon Girl, Lil’ Pump Ke Mon, Spaghetti) of seedlings and six cultivars (Cinnamon Girl, Jack Be Little, Lil’ Pump Ke Mon, Honeyboat, Spaghetti, Sweet Dumpling) of fruit. gyrB sequence data of isolates recovered after inoculation were comparable to PsAs_2015 on 33 to 100% of seedlings and fruit. The clustering of PsAs_2015, PsFt1_2015, and PsFt2_2015 with other genomospecies 1 pathovars confirms that they are distinct from P. syringae pv. lachrymans and represents a first report for Washington State. Moreover, the presence of wart symptoms on pumpkin fruit represents a newly observed symptom in the United States.

FIRST REPORT OF HYBRIDIZATION BETWEEN TWO FORMAE SPECIALES PUCCINIA STRIIFORMIS f. sp. HORDEI AND PUCCINIA STRIIFORMIS f. sp. TRITICI IN IRAN

During 2012-2013, yellow rust samples from barley, wild barley (Hordeum morinum) and wheat from different provinces of Iran were used to inoculate Afzal (barley cultivar) and Morocco (wheat cultivar), susceptible to all Puccinia striiformis f. sp. hordei (Psh) and Puccinia striiformis f. sp. tritici (Pst) isolates, respectively. The isolates were identified as Psh or Pst based on virulence on barley or wheat differential seedlings, respectively (Chen et al., 1995). In order to study of genetic diversity, we used 18 isolates representative of different pathotypes from different hosts. The genetic diversity was assessed using five simple sequence repeat (SSR) markers and by examining intergenic spacer 1 (IGS1) region. A total of 14 genotypes were identified within the studied isolates, of which eight genotypes related to Psh and six genotypes related to Pst. The multiplied fragments of some SSR loci, in particular RJ2N, showed that some isolates have alleles in common with isolates of Psh and Pst. Dendrograms were generated using virulence and markers data. Clustering analysis showed groups related to their hosts: isolates infecting only wheat, isolates infecting only barley; isolates infecting both wheat and barley differential genotypes; and isolates infecting only wild grasses with some overlaps. The groups infecting both barley and wheat differentials (based on virulence data) and having some SSR alleles in common with Psh and Pst, appeared to be hybrid group between two formae speciales. Before this report, Cheng and Chen (2009) and Holtz et al. (2014) had reported hybridization between two formae speciales.

Pathotypes of Pyrenophora teres on barley in Turkey

Net blotch foliar diseases of barley are important in Turkey, lowering grain yields and quality. There are two forms, the spot form (caused by Pyrenophora teres f. maculata ( Ptm )) and the net form (caused by P. teres f. teres ( Ptt )) . To determine the pathotypes of Ptt and Ptm in Turkey, surveys were carried out during 2012, 2013 and 2015. Pyrenophora teres samples were collected from 34 provinces of Turkey. From these samples, 258 Ptm and 167 Ptt single conidium isolates were obtained. Pathotypes of 50 P. teres f. maculata and 40 P. teres f. teres isolates were assessed by inoculating onto a differential set of 25 barley genotypes. Twenty six Ptm pathotypes and 24 Ptt pathotypes were identified, and significant pathogenic variation was found among the isolates. Barley breeding programmes in Turkey should consider the pathotypes identified for incorporation of net blotch resistance. Continuous virulence monitoring for the P. teres population should be carried out to inform resistance breeding priorities.

Bacterial pathogens of pigs with particular reference to Escherichia coli: A systematic review and meta-analysis

Pigs are ungulate animals of the genus Suis and family Suidae. They are globally spread but restricted in certain countries due to religious and cultural beliefs. Pork serves as an important source of protein (38% of meat consumed in the world). While pig production remains a profitable enterprise, commercial and particularly the small-scale farmers face huge constraint in this husbandry practice, one of the most important being bacterial infections and its associated with morbidity and mortality. In this work, we reviewed the prevalence of bacterial infections in pigs with particular reference to Escherichia coli, a bacterium that is regularly isolated and can lead to multiple infections in pigs. Literatures were searched on selected veterinary and biological data bases in 2016 with focus on natural infections and isolates from natural infections with epidemiological details. Pathotypes, serotypes and serogroups of E. coli, the country of origin, source, growth stage, age of pigs infected, disease outbreak, the number of samples and type of samples, numbers and percentage of positive samples and isolates were used as filters. Pathotypes reported include enterotoxigenic E. coli (ETEC) 66.7%, enterotoxigenic E. coli and shiga toxigenic E. coli (ETEC and STEC) 14.3%, STEC only (7.9%), enterotoxigenic E. coli/enteropathogenic E. coli/enteroaggregative E. coli (ETEC/EPEC/EAE) 31.7%. Others were enterohaemorrhagic E. coli (EHEC), diffusely adherent E. coli (DAEC) (ETEC, EPEC, STEC) and extra-intestinal pathogenic E. coli (ExPEC). Twenty-nine countries with documented records of cases of E. coli were included with the USA reporting, the highest number followed by China. About 74% of the samples were taken from farms and others were from samples submitted to research laboratories and veterinary faculties for necropsy. Serogroups O141, O149, O139, O138, O8 and O9 were most common. Piglets were most affected (52.3%) followed by weaners (39.6%) and porkers (7.9%) with age ranging from 1 to 392 days old. A total of 24,854 isolates were considered, 10477 (42.2%) were positives and the following genes were carried: STa, STb, LT, stx1, stx2, Stx-2e, F4, F5, F6, F18, F41, AIDA, EAST1, eae, paa and hlyA. The diseases produced by E. coli were neonatal diarrhoea, colibacillosis, post-weaning diarrhoea and edema disease. The associated risk factors were poor housing, management and feed changes, extensive use of antibiotics as prophylaxis, overcrowding, and high humidity and temperature changes. India, USA, Japan, Slovakia, Denmark Sweden and Poland were countries with significant reports and high detection of virulence factors (72 to 100%). Key words: Escherichia coli, diarrhea, serogroups, enterotoxigenic, colibacillosis.

Detection of specific antigens of Newcastle disease virus using an absorbed Western blotting method.

Newcastle disease virus (NDV) is an economically important poultry pathogen with a worldwide distribution that may infect a wide range of domestic and wild avian species. The identification of different pathotypes of NDVs plays an important role in the diagnosis and development of vaccines to control and eradicate NDV infections. In our previous study, we showed that mono-specific antibodies can differentiate velogenic and lentogenic strains of NDV in Agar Gel Immuno-Diffusion tests. To evaluate the ability of the specific antibodies to detect NDV specific antigens, this study was conducted with a range of NDV isolates. The samples included 9 NDV neuropathogenic/velogenic isolates from diseased chickens collected from poultry farms in central and northern parts of Iran plus La-Sota and B1 vaccine strains. All samples were propagated in embryonated chicken eggs and concentrated and purified by ultra-centrifugation. All samples were subjected to 12.5% SDS-PAGE and Western blotting using the specific antibodies mentioned previously. In SDS-PAGE all velogenic and vaccine strains showed the same electrophoretic pattern. The detected bands included 15, 38, 46, 48, 53, 55, 68, 74 and 220 kDa proteins. In Western blotting analysis, the mono-specific antibodies reacted specifically to the viral proteins with 15, 38, 48, 55, 74 and 220 kDa and non-specifically to the viral protein with 53 kDa. The results suggest that specific anti-NDV antibodies can react specifically to glycoproteins (haemagglutin-neuraminidase and fusion proteins) but not to internal proteins (nucleoprotein or matrix protein) of NDV strains.

DIVERSITY OF IRANIAN STRAINS OF PSEUDOMONAS SYRINGAE pv. SYRINGAE AND PSEUDOMONAS SYRINGAE pv. LACHRYMANS

Strains of Pseudomonas syringae pv. syringae (Pss), Pseudomonas syringae pv. lachrymans (Psl) and Pseudomonas tolaasii (P. tolaasii) were analyzed for genetic diversity. For molecular identification of the strains, three primer pairs were used. Molecular analysis was done with random amplified polymorphic DNA (RAPD) and Insertion Sequence (IS50) primers. IS50-PCR and RAPD-PCR analysis divided 51 strains into eight sub clusters that were grouped in three clusters. Cluster I comprised all Pss with four sub clusters from stone fruits, pome fruit and cereal hosts, cluster II contained Psl strains from cucumber with two sub clusters and cluster III contained three strains of P. tolaasii with two sub clusters from mushroom. These results indicated that a combination of IS50-PCR and RAPD-PCR fingerprinting can be used as a high resolution genomic fingerprinting methods for differentiates species of Pseudomonas and pathovars of P. syringae strains.

DETECTION OF XANTHOMONAS CITRI subsp. CITRI A* , AW AND X. FUSCANS subsp. AURANTIFOLII B, C USING PCR AND REAL-TIME PCR

Citrus bacterial canker (CBC) is an economically important disease in many citrus producing regions, and several canker-causing pathotypes have been characterized within the genus Xanthomonas pathogens. In view of the potential for economic losses and the quarantine significance of the pathogens, rapid and reliable pathogen diagnostic methods are needed for controlling this disease. In this study, we report novel molecular markers for Xanthomonas strain specific detection by polymerase chain reaction (PCR). The primer sets XcAsF/XcAswR, XaBF/XaBR, and XaCF1/XaCR1 specifically differentiated Xanthomonas citri subsp. citri A* and Aw; X. fuscans subsp. aurantifolii B; and X. f. subsp. aurantifolii C strains, respectively. In addition, real-time PCR (qPCR) assays were conducted to analyze the specificity and efficiency of the designed primers and also their sensitivity by estimating the minimum quantity of bacterial cells required for detection. With qPCR analysis, it was possible to detect a range of one to 1 × 107 bacterial cell(s), which will be helpful to detect the species at pre-symptom development stage of quarantine period of the plant. PCR/primer efficiency was demonstrated also by using raw bacterial cells, which will reduce considerable time and cost needed for genomic DNA isolation. The identified molecular markers and qPCR method will be useful for routine diagnosis of Xanthomonas species, which will improve the citrus canker disease management.

Genetic diversity of Xanthomonas citri subsp. citri in citrus orchards in northwest Paraná state, Brazil

Xanthomonas citri subsp. citri , which causes Asiatic citrus canker (ACC), is an important pathogen of citrus in Brazil and elsewhere. The genetic diversity of X. citri subsp. citri pathotype ‘A’ has not been studied in Brazil at a local scale (up to 300 km). Forty isolates were sampled from lesions of ACC on citrus in three orchards in Parana state, Brazil. Twelve minisatellite markers were used to characterize the genetic diversity of the isolates. An Unweighted Paired Group Method of Arithmetic Averages tree was used for identifying unique multilocus haplotypes but there was no association between haplotypes and source locations. An analysis of molecular variance among populations showed that 98% of the variance was accounted for within the populations, and only 2% was accounted for among populations. Differences among populations was not significant (Φ=0.018, P =0.2). The relatively high, yet uniform, genetic diversity among isolates and low degree of spatial differentiation between populations of X. citri subsp. citri suggests that the populations in Parana state have a common origin and strong historical epidemiological links.

FIRST REPORT OF CITRUS DEPRESSA AS A NEW NATURAL HOST OF XANTHOMONAS CITRI subsp. CITRI PATHOTYPE A IN TAIWAN

Citrus depressa is an indigenous citrus species in Taiwan. Not much is known about diseases occurring on C. depressa. A leaf spot disease of C. depressa was first observed in Chiayi City, Taiwan, in 2015 and 2016. Symptoms were brownish necrotic spots, surrounded by a yellowish halo. Leaf blight was not observed. Bacteria were isolated from surface-sterilized leaf spot samples, and two Xanthomonas-like yellow isolates, CJHCd003 and CJHCd004, were purified and stored at -80°C in 20% glycerol. Leaves of C. depressa, sweet orange (C. sinensis cv. Liucheng), mandarin (C. reticulata cv. Ponkan), grapefruit (C. paradisi), and Mexican lime (C. aurantiifolia) were infiltrated with bacterial suspensions (1×106 CFU/ml). C. depressa inoculated leaves exhibited necrotic spots surrounded with a yellowish halo ten days post inoculation similar to those observed in natural infections. The same bacterium could be consistently re-isolated from the inoculated leaves and confirmed by PCR with 4/7 primers (Hartung et al., 1993), fulfilling Koch’s postulate. The control leaves remained disease-free. Multilocus sequence analysis of seven housekeeping genes (16S rDNA, fusA, gap-1, gltA, gyrB, lacF, and lepA) (Almeida et al., 2010; Ngoc et al., 2010) fully identified the two isolates from C. depressa as pathotype A strains of X. citri pv. citri with 100% sequence identity (Ngoc et al., 2010; Zhang et al., 2015). Furthermore, these strains caused typical erumpent, callus-like tissue at inoculated sites on the other citrus leaves tested, indicating that these strains belong to pathotype A strains of X. citri subsp. citri. To our knowledge, this is the first report of C. depressa as a newly discovered natural host of X. citri subsp. citri pathotype A in Taiwan.

Inheritance study of blast resistance with pathotype P7.2 on crosses of Pongsu Seribu 2 and MR264

Inheritance study of blast resistance with pathotype P7.2 on crosses of Pongsu Seribu 2 and MR264

SCOPOLIN, A GLYCOSIDE FORM OF THE PHYTOALEXIN SCOPOLETIN, IS LIKELY INVOLVED IN THE RESISTANCE OF NICOTIANA ATTENUATA AGAINST ALTERNARIA ALTERNATA

Phenolic coumarins are secondary metabolites playing an important role in plant-environment interaction. Previously, we have shown that wild tobacco Nicotiana attenuata accumulates high amounts of the jasmonate (JA)-dependent phenolic coumarin, scopoletin, as a phytoalexin to defend itself against a necrotic fungal pathogen, Alternaria alternata (tobacco pathotype). Scopolin, a s-glycoside form of scopoletin, was strongly elicited and to a level even higher than that of scopoletin in host plants after A. alternata infection. However, it was not known whether scopolin functioned as a phytoalexin playing a role in the resistance or simply as a storage form of scopoletin. When supplied in PDA medium, scopolin exhibited a moderate but slightly weaker anti-fungal activity against A. alternata than that of scopoletin. In addition, A. alternata-elicited scopolin level was dramatically decreased in JA deficient plants. Thus, all the data suggest that scopolin is also a JA-dependent phytoalexin likely involved in the resistance of N. attenuata against A. alternata.

OCCURRENCE OF CUCUMBER MOSAIC VIRUS IN OLEANDER (NERIUM OLEANDER) IN HUNGARY

In June 2016 mosaic symptoms characteristic of viral in- fection were observed in a ten year-old potted oleander ( Ner- ium oleander ) maintained in Velence (Hungary). Inoculation of test plants ( Chenopodium , Cucumis and Nicotiana spp.) with extracts of diseased oleander leaves demonstrated the presence of a mechanically transmitted plant virus marked Nol. The Nol isolate caused local lesions on Chenopodium quinoa , mosaic on Cucumis sativus and local etched ring pat- tern followed by vein clearing and recovery on the top leaves of Nicotiana tabacum cv. Xanthi-nc. These symptoms sug- gested the presence of B pathotype (subgroup II strain) of Cucumber mosaic virus (CMV). CMV was identified in the oleander leaves using CMV specific lateral flow immunoas- says (Bioreba AgriStrip). To confirm the presence of CMV, t otal RNA was extract- ed from N . clevelandii infected with CMV-Nol and used in RT-PCR with subgroup I and II CMV coat protein (CP) gene specific primers, respectively (Salanki et al ., 1997). The DNA fragment of the expected size (1460 bp) was amplified from tobacco leaves using subgroup II specific primers solely. The PCR product was cloned into pGEM-T Easy Vector (Pro- mega, USA), sequenced (Biomi, Hungary) and the complete CP gene nucleotide sequence was deposited in GenBank (accession No. KX951465). The complete CP gene of CMV- Nol showed 99% nucleotide and 100% amino acid sequence identity with a Serbian CMV isolate (232Mrg, accession No. KP034963.1). This is the first record on the natural occur- rence of CMV in oleander in Hungary.

FIRST REPORT OF A NEW PEPPER MILD MOTTLE VIRUS PATHOTYPE 1,2 ON PEPPER IN SPAIN

During the summer of 2015, leaf deformations and chlorotic mosaic symptoms on leaves and unusual necrotic lesions on leaves, stems and fruits were observed on pepper (Capsicum annuum) plants grown in Almeria (Spain). Symptomatic leaf and fruit samples from eight collected plants, carrying L3 and L4 resistance against tobamovirus, were analyzed by ELISA specific for potyviruses, Cucumber mosaic virus and each of the following possible pepper-infecting viruses: Paprika mild mottle virus, Tobacco mosaic virus, Tomato mosaic virus, Tobacco mild green mosaic virus, and Pepper mild mottle virus (PMMoV). The eight plants tested negative for all viruses except for PMMoV. Mechanically inoculated extracts from two PMMoV-positive, singly infected symptomatic pepper plants systemically infected C. annuum cv Mazo (Unigenia), but not C. chinense PI159236, C. chacoense PI260429, four commercial L4, and two L3 resistant pepper plants, which exhibited local necrosis symptoms. Total RNAs from two plants inoculated with single infections were extracted, analyzed by reverse transcription PCR using primers designed to specifically amplify the coat protein gene (CP) of PMMoV (Antignus et al., 2008) and directly sequenced. Two independent samples yielded identical nt sequences (GenBank accession No. KT962848) and BLAST analysis showed the highest deduced aa identities of 99.4% with P1,2 isolates (M81413, AB000709, EF434393) of PMMoV. However, the predicted aa sequence possessed an Ala→Thr mutation at residue 66 of the CP. To our knowledge, although PMMoV has been shown to have variants that fail to infect L3 and L4 resistant pepper plants, based on unique CP coding sequences, this is the first report of a new P1,2 pathotype in Spain.

BIOLOGICAL AND MOLECULAR CHARACTERISTICS OF BEAN COMMON MOSAIC VIRUS ISOLATES CIRCULATING IN COMMON BEAN IN IRAN

Bean common mosaic virus (BCMV) is one of the most widespread and economically important seed and aphid-transmitted viruses. During 2010-2011, a total of 2410 symptomatic bean (Phaseolus vulgaris) leaf samples were collected from nine provinces of Iran and checked by DAS-ELISA (double antibody sandwich enzyme-linked immunsorbent assay), resulting in detection of BCMV in 655 samples. Following biological purification, the reaction of 16 differential bean cultivars (belonging to 11 host groups) against five geographical BCMV isolates revealed the occurrence of five different BCMV pathogroups, PG- I, II, IV, V and VII, which were new for Iran. The response of five commonly cultivated bean genotypes against three BCMV pathotypes (I, II and IV) was evaluated by semi-quantitave ELISA and two genotypes, Chiti karaj and Jules, were characterized as highly sensitive and tolerant genotypes, respectively. Pathogenicity of three different BCMV pathotypes on five bean genotypes used in this study revealed that they all belong to host group-1. Amplification of the 3'-terminal region (comprising coat protein region and partial 3'-UTR) of four BCMV isolates was performed by reverse transcription polymerase chain reaction (RT-PCR). Phylogenetic analysis of nucleotide and deduced amino acid sequences of coat protein genes did not differentiate BCMV isolates by host, location and pathogenicity group.

SOFT ROT OF ONION BULBS CAUSED BY BURKHOLDERIA GLADIOLI PV. ALLIICOLA IN SLOVENIA

In 2012, growers of an indigenous onion (Allium cepa L.) cultivar from Northeastern Slovenia began noticing an increase of rotting onion bulbs both in the field and in storage. In some instances up to 30% of the bulbs were affected. Externally, the bulbs appeared healthy, but internal layers were found to have water-soaked and brown coloured lesions. We obtained samples from two different onion storage locations in spring of 2013 and isolated 27 morphologically distinct bacterial colonies on King’s medium B. The isolates were identified based on 16 S rDNA sequences (Bandi et al., 1994). An identical 16 S rDNA sequence (GenBank accession No. LT547718) was obtained from four isolates found at both storage locations and identified as a potential onion pathogen, Burkholderia gladioli based on high sequence similarity to other B. gladioli sequences (100 %) in the database. The strains of B. gladioli exhibited two different colony morphologies. One had a slightly wrinkled surface while the other was smooth. Both exhibited typical white-yellowish colour and released a yellow diffusive pigment into the medium. Pathogenicity of B. gladioli strains was evaluated on the onion bulbs by injecting a bacterial suspension into the bulb’s neck. Only B. gladioli strains caused a light brown rot development on the bulb’s internal layers after 6 to 12 days at 25oC. These results suggested the isolated B. gladioli strains belonged to the pathovar alliicola which was recently associated with extensive onion rotting in Poland (Kowalska et al., 2015). This is the first report and identification of Burkholderia gladioli pv. alliicola as a causative agent of soft rot on onion in Slovenia.

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF TOBACCO MILD GREEN MOSAIC VIRUS IN HUNGARY

Pepper (Capsicum annuum L.), an important crop in Hun- gary, is frequently infected by Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV) and, recently, also by Pepper mild mottle virus (PMMoV) and Tomato spotted wilt virus (TSWV). In the spring of 2015, mild mosaic symptoms were observed on cv. Ho F1. Symptoms on the fruits were more obvious, consisting of reduction in size, mottling and color changes, brown necrotic streaks and spots. To identify the putative pathogen(s), symptomatic fruits were collected and used for host range determination. Symptoms on mechani- cally inoculated Nicotiana tabacum cv. Samsun, N. tabacum cv. Xanthi-nc, Nicotiana benthamiana, Capsicum annuum cv. Albaregia (L+ gene), C. annuum cv. Feherozon (L1) and C. annuum cv. Brendon F1 (L3) suggested the presence of a tobamovirus belonging to pathotype 0 (Boukema, 1980). Moreover, total RNA was extracted from a symptomatic pepper fruit with the RNeasy plant mini kit (Qiagen, Ger- many) and used in RT-PCR using universal tobamovirus primers for the coat protein gene (Kalman et al., 2001). A products of the expected size (700 bp) was obtained, cloned into pGEM-T Easy Vector (Promega, USA) and sequenced (Biomi, Hungary). The sequence was deposited in GenBank under the accession No. KT374283. Blast analysis showed a 99% identity at nucleotide level with a Spanish isolate (P04/17, accession No. FN594859) of Tobacco mild green mosaic virus (TMGMV). To our knowledge, this is the first report of TMGMV on pepper in Hungary.