Journal of Plant Pathology (2015), 97 (1), 37-43 Edizioni ETS Pisa, 2015 37

BURKHOLDERIA GLADIOLI ASSOCIATED WITH SOFT ROT OF BULBS IN POLAND

B. Kowalska, U. Smoli ´nska and M. Oskiera

Research Institute of Horticulture. Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland

SUMMARY (Gitaitis et al., 1998), Pantoea ananatis (Gitaitis et al., 2002; Walcott et al., 2002), Enterobacter cloacae (Schroeder et al., Bacterial diseases of onion (Allium cepa L.) are serious 2009), ambifaria and B. pyrrocinia (Jacobs et problems in Poland. In this study bacterial strains were al., 2008) and Serratia spp. (Beriam, 2007) have also been isolated from onion bulbs with soft-rot symptoms. Patho- reported as agents of onion bacterial diseases. genicity tests were conducted on onion bulbs and also on B. gladioli pv. alliicola was first reported as Phytomo- tobacco leaves on whose basis twelve isolates were chosen nas alliicola from rotten onion bulbs in New York state to further characterization. These isolates were identified (USA) (Starr and Burkholder, 1942); then, under the name using physiological and biochemical tests, and confirmed of gladioli, from Iowa (USA), as the cause by species-specific PCR, ERIC-PCR and sequence analysis of yellowing and death of onion leaves and infection of of 16S rRNA, gyrB, lepA, phaC, recA gene fragments. All the outer scales of young bulbs (Semeniuk and Melthus, examined isolates were identified as . 1943). The bacterium was isolated from onion bulbs by It is the first report of the occurrence of B. gladioli on both Burkholder and Vitanov (Burkholder, 1950) who re- onion as the cause of onion disease in Poland. garded it exclusively as an onion bulb pathogen and was reclassified as Burkholderia gladioli (Yabuuchi et al., 1992). Key words: Burkholderia gladioli, onion, diagnosis, soft Its presence in onion has been reported from Europe, rot, disease. Asia and USA (Lee et al., 2005; Schwartz and Mohan, 2008; Stoyanova et al., 2011; Schroeder et al., 2012), with crop losses of up to 40%. INTRODUCTION B. gladioli strains have also been isolated from and re- garded as a pathogen of sp., sp., Eustoma Onion (Allium cepa L.) is one of the major vegetable grandiflorum (Coenye and Vandamme, 2003; Stoyanova et crops grown in Poland, where the total area planted and al., 2007; Keith et al., 2005), summer snowflake (Leucojum harvested in 2012 was ca. 25,000 ha. Since onion harvest- aestivum) (Stoyanova et al., 2013), saffron (Crocus sativus L.) ing often coincides with rainy weather and during cultiva- (Fiori et al., 2011), maize (Lu et al., 2007; Gijon-Hernandez tion hailstorms may occur, complex bacterial and fungal et al., 2011) and rice (Ura et al., 2006; Nandakumar et al., diseases often develop. In recent years, bacterial diseases 2009). have caused very serious problems to Polish onion crops, B. gladioli can also be a human pathogen, but it can inflicting significant economic losses because they are dif- hardly be identified with commercial detection kits. Its ficult to control. Successful control depends on proper strains are sensitive to the complement-mediated lysis sanitation, avoiding injuries, keeping bulbs dry and cool of human serum, which confers a natural immunity to during storage, assuring good insect control and prac- healthy individuals. However, one of the four cases of hu- ticing crop rotation (Agrios, 2005). Bacterial soft rot of man infection was in a non-immunocompromised patient onion bulbs is most frequent and it can appear during (Stoyanova et al., 2007). cultivation, storage or transportation (Sobiczewski and Very little is known on B. gladioli occurrence in Poland Schollenberger, 2002). and the characteristics of its strains. There are only a few Soft-rot diseases of bacterial origin, particularly those short reports on onion soft rot induced by Burkholderia sp. associated with Burkholderia gladioli, B. cepacia, Pectobac- (Sobiczewski and Schollenberger, 2002), but no informa- terium carotovorum subsp. carotovorum (Schwartz and tion on the biochemical and molecular properties of the Mohan, 2008; Yohalem and Lorbeer, 1997), Serratia plymu- associated , except for a paper by Schollenberger thica (Kowalska et al., 2011) are known all over the word. and Zamorski (2008) who observed atypical disease symp- Pseudomonas marginalis (Kim et al., 2002; El-Hendawy, toms on lisianthus growing in greenhouses near Warsaw. 2004), Pseudomonas syringae, Pseudomonas viridiflava The bacterium isolated from diseased plants was identi- fied as B. gladioli by conventional microbiological meth- Corresponding author: B. Kowalska Fax: +48.46.8333186 ods, and the disease was named bacterial ring blight of E-mail: [email protected] lisianthus (Schollenberger and Zamorski, 2008). 38 Burkholderia gladioli in Poland Journal of Plant Pathology (2015), 97 (1), 37-43

Table 1. Bacterial strains used in this study and GeneBank accession Nos.

GenBank accession No. Bacterial isolates Locality, place and year of isolation gltB lepA phaC recA Bg259 Poland, Tomaszów Lubelski; field; 2006 - KF857500 KF857513 KF857525 Bg260 Poland, Tomaszów Lubelski; field; 2006 KF857489 KF857501 - KF857526 Bg261 Poland, Tomaszów Lubelski; field; 2006 KF857490 KF857502 KF857514 KF857527 Bg285 Poland, Radom; field; 2006 KF857491 KF857503 KF857515 - Bg294 Poland, Radom; field; 2006 KF857492 KF857504 KF857516 KF857528 Bg295 Poland, Radom; field; 2006 KF857493 KF857505 KF857517 KF857529 Bg435 Poland, Skierniewice; storage; 2007 KF857494 KF857506 KF857518 KF857530 Bg469 Poland, Skierniewice; storage; 2007 - KF857507 KF857519 KF857531 Bg467 Poland, Skierniewice; storage; 2007 KF857495 KF857508 KF857520 KF857532 Bg494 Poland, Skierniewice; field; 2008 KF857496 KF857509 KF857521 KF857533 Bg500 Poland, Skierniewice; field; 2008 KF857497 KF857510 KF857522 KF857534 Bg514 Poland, Hopkie; storage; 2008 KF857498 KF857511 KF857523 KF857535 B. gladioli pv. alliicola LMG 6979 USA KF857488 KF857499 KF857512 KF857524 B. cepacia LMG 6962 USA - - - -

The objective of this study was the identification of each piece was wounded with a laboratory needle to make bacteria associated with soft rot of onion in Poland, using a wound 3-4 mm in diameter which was inoculated with biochemical and molecular methods. 20 µl suspension from a 24 h pure bacterial culture con- taining 1.0-2.5 × 108 CFU ml−1. Controls were not inoculat- ed. Inoculations of reference strains B. cepacia LMG 6962 MATERIALS AND METHODS and B. gladioli LMG 6979 were also made consisting of two replicates (six onion pieces) for each bacterial strain. Isolation of soft rotting bacteria. Onion bulbs with Disease symptoms were examined after 4 days incubation bacterial soft rot were obtained during summer and au- at 28°C. Virulence was evaluated using an arbitrary scale tumn of 2006, 2007 and 2008 from Polish onion storage from 0 (no tissue maceration) to 3 (complete maceration). warehouses or field crops. The bulbs were washed with tap The experiment was repeated twice. For the experiments water and cut lengthwise. Diseased scale tissues were then that followed only the isolates were used that induced mac- cut into about 10-15 mm cubes with a sterilized scalpel, the eration of level two and greater than two. A total of 12 fragments were sterilized in ethanol for 30 sec, washed in isolates were investigated: Bg259, Bg260, Bg261, Bg285, sterile water and plated onto nutrient agar medium (NA, Bg294, Bg295, Bg435, Bg467, Bg469, Bg494, Bg500 and beef extract 3 g, glucose 2.5 g, peptone 5 g, agar 15 g, and Bg514 (Table 1). distilled water 1000 ml). Petri dishes with NA were incu- Tobacco hypersensitivity reaction was conducted us- bated at 28°C for 48 h. Next, a selected bacterial colony ing tobacco plants of cv. Samsun (Klement et al., 1964). A was streaked onto a fresh NA plate and used for this study. bacterial suspension of 1.0-2.5 × 108 CFU ml−1 was injected into the intercellular space of tobacco leaves, recording as Bacterial strains and culture conditions. Forty two positive the complete collapse of the tissues after 24 h. The bacterial isolates used in this study were maintained at test was repeated with each isolate at least twice. −80ºC in nutrient broth medium containing 50% (v/v) glycerol. The isolates were transferred onto NA, incubat- Biochemical and physiological tests. For these assays ed at 28°C for 24 h and maintained at 4°C for short-term 12 bacterial isolates were used, that gave positive responses use. Genuine isolates of B. gladioli pv. alliicola LMG 6979 in pathogenicity tests. Bacterial colony morphology was and B. cepacia LMG 6962, provided by from a Belgian assessed on NA medium taking into account the shape, Microorganism Collection (Ghent University) were used size, texture and markings of the surface. Physiological for comparison. and biochemical characterization was according to Schaad et al. (2001). The following properties were determined: Pathogenicity tests. All strains were examined for Gram reaction by 3% KOH (Suslow et al., 1982) and the ability to macerate onion tissue. To this aim, healthy Gram stain, anaerobic growth in Hugh and Leifson (1953) bulbs of cv. Grabowska were peeled, washed with run- medium, production of fluorescent pigment on King B ning water and sterilized in 70% ethanol for 30 sec and medium [peptone 20.0 g, K2HPO4 2.5 g, glycerol 15 ml, in 0.5% NaOCl for 5 min. The bulbs were then washed MgSO4 × 7H2O 6.0 g, agar 15.0 g, water 1000 ml (King et in sterile water and cut lengthwise into two parts. Three al., 1954)]; colony colour on YDC medium [yeast extract onion pieces were placed into a large Petri dish incuba- 10.0 g, glucose 20.0 g, CaCO3 20.0 g, agar 15.0 g, water tion chamber (volume 314 cm3), on a paper filter which had 1000 ml (Wilson et al., 1967)]; growth on D1M medium been wetted with 10 ml of sterile water. The outer scale of [cellobiose 5.0 g, NH4Cl 1.0 g, NaH2PO4 1.0 g, K2HPO4 Journal of Plant Pathology (2015), 97 (1), 37-43 Kowalska et al. 39

Table 2. PCR primers used for Burkholderia spp. identification.

Gene Primer name Sequence (5’→ 3’) Reference Specific for B. gladioli 16S rRNA CMG-16-1 = fD1 AGAGTTTGATCMTGGCTC Weisburg et al., 1991 G-16-2 CGAAGGATATTAGCCCTC Specific for B. gladioli 23S rRNA CMG-23-1 ATAGCTGGTTCTCTCCGAA Bauernfeind et al., 1998a, 1998b G-23-2 CCTACCATGCAYATAAAT ERIC-PCR genome ERIC1R ATGTAAGCTCCTGGGGATTCAC DNA “fingerprint” Louws et al., 1995 ERIC2 AAGTAAGTGACTGGGGTGAGCG

1.0 g, MgSO4 × 7H2O 3.0 g, malachite green 10.0 mg, agar different PCR thermal profile was used: 5 min denatur- 15.0 g, water 1000 ml (Perry and Kado, 1982]); utilization ation at 95°C, 35 amplification cycles of 1 min at 94°C, of arginine [peptone 5.0 g, yeast extract 5.0 g, K2HPO4 90 sec at 52°C and 8 min at 65°C, followed by a final ex- 2.0 g, dextrose 50.0 g, arginine HCl 3.0 g, water 1000 ml tension step of 16 min at 65°C. Amplified products were (Cowan, 1974)]; growth at 40°C; oxidase reaction. Oxidase checked by 1.5% agarose (Basica Le Gqt, Prona, Norway) test was conducted using a filter paper impregnated with gel electrophoresis and stained with ethidium bromide 1% aqueous tetramethyl-p-phenylenediamine dihydrochlo- (0.25 µg ml−1). ride solution (Fluka, Germany). A small loopful of the in- The identity of the bacterial isolates was confirmed by oculum was transferred onto the filter paper. The isolate using Burkholderia-specific PCR primers (Bauernfeid et was rated oxidase-positive if a purple colour developed al., 1998a, 1998b) (Table 2). ERIC-PCR primers (Louws within 10 sec, negative if no colour developed after 60 sec. et al., 1995) were used to generate and compare genomic Utilisation of the following carbohydrates: lactose, su- fingerprints of bacterial isolates and B. gladioli pv. alliicola crose, sorbitol and cellobiose was investigated. All carbon strain LMG 6979. Primers ERIC1R and ERIC2 (Table 2) sources were added at 0.1% final concentration to a min- correspond to conserved motifs in bacterial repetitive se- eral salts medium consisting of K2HPO4 7 g, KH2PO4 quences. Sequencing of the 16S rRNA, gyrB, lepA, phaC, 2 g, MgSO4 × 7H2O 0.1 g and (NH4)2SO4 1 g per liter of recA gene fragments was done with the PCR primers listed distilled water (pH 7). The medium was filter-sterilized in Table 3. Amplicons were generated with the same prim- and solidified with agar. Bacteria were streaked onto the ers as used for further sequencing in both directions. Only plates which were incubated at 28°C. The growth was ob- three representative isolates Bg259, Bg295, and Bg494 were served over seven days. Reference bacteria were used as a chosen for 16S rRNA gene sequencing. 16S rRNA ampli- standard. cons were generated and sequenced with fD1, rP2 primers The ability of the bacteria under study to grow on se- (Weisburg et al., 1991) and additionally primers 800f and lective PCAT and CB media was also examined. PCAT 800r (Drancourt et al. 1997) were used for sequencing. medium consisted of azelaic acid 2 g, tryptamine 0.2 g, PCR conditions used for amplification were as above for MgSO4 × 7H2O 0.1 g, K2HPO4 4 g, KH2PO4 4 g, yeast ex- standard PCR without modifications. PCR products were tract 0.02 g, agar 15 g, water 1000 ml and chlorothalonit cleaned with Exo-Sap (Affymetrics, USA) procedure fol- 1 ml added after sterilization (Salles et al., 2006). CB me- lowing manufacturer’s instructions and 6 µl total mixture dium consisted of tryptone 5 g, yeast extract 2.5 g, glucose of 20 ng PCR amplicons with 5 µM primer were sequenced 1 g, agar 15 g, water 1000 ml and polymyxine B sulfate (Genomed, Poland). Editing and analysing of the obtained added after sterilization (Wu and Thompson, 1984). sequences were performed with CLC Genomic Work- bench 6 and NCBI megablast software. DNA-based techniques. For molecular analysis, total genomic DNA was isolated according to Aljanabi and Martinez (1997). Amplification reactions were performed RESULTS AND DISCUSSION in a 20 µl final reaction mixture containing 1× PCR buffer (with 2 mM MgCl2, 250 µM of each deoxynucleoside tri- A bacterium responsible for soft rotting was consis- phosphate 0.5 µM of each primer, 1 U of DreamTaq DNA tently isolated from diseased in the field and from polymerase for standard PCR or Taq DNA polymerase (all bulbs under storage. During storage, infected onion bulbs by Thermo Scientific, USA) for ERIC-PCR and 20 ng of did not show any symptoms except for softening of the bacterial DNA. PCR was performed with Mastercycler ep neck tissue but, when cut longitudinally, one or two in- gradient S (Eppendorf, USA) under the following condi- ner fleshy scales appeared soft and water-soaked or pale tion: denaturation for 3 min at 95°C, then 35 amplification brown. Eventually the infection spread to other scales and cycles of 30 sec at 94°C, 30 sec at the appropriate annealing in the more severe cases, soft rotting of the internal scales temperature and 60 sec at 72°C and followed by a final extended to the external ones which emanated a sour extension step of seven min at 72°C. For ERIC-PCR a smell, turned brown and dried out. 40 Burkholderia gladioli in Poland Journal of Plant Pathology (2015), 97 (1), 37-43

Table 3. Primers used for sequencing of gene fragments and amplicons generation, with exception of Drancourt’s primers which were used for sequencing reactions only.

Gene Primer name Sequence (5’→ 3’) References 16S rRNA gene almost full length fD1 AGA GTT TGA TCM TGG CTC Weisburg et al., 1991 rP2 ACGGCTACCTTGTTACGACTT 16S rRNA gene half length 800f ATTAGATACCCTGGTAG Drancourt et al., 1997 800r CTACCAGGGTATCTAAT gyrB (DNA gyrase subunit B, partial sequence) gyrB for ACCGGTCTGCAYCACCTCGT

gyrB rev YTCGTTGWARCTGTCGTTCCACTGC lepA (GTP binding protein, partial sequence) lepA for CTSATCATCGAYTCSTGGTTCG lepA rev CGRTATTCCTTGAACYCGTARTCC Spilker et al., 2009 phaC (phaC gene partial sequence) phaC for GCACSAGYATYTGCCAGCG phaC rev CCATSTCSGTRCCRATGTAGCC recA (recombinase A, partial sequence) recA for AGGACGATTCATGGAAGAWAGC recA rev GACGCACYGAYGMRTAGAACTT

Onion bulbs artificially inoculated with bacterial isolates compound playing a role in the virulence of B. gladioli to Bg259, Bg260, Bg261, Bg285, Bg294, Bg295, Bg435, Bg467, rice seedling (Ura et al., 2006). Bg469, Bg494, Bg500 and Bg514 developed symptoms of All studied isolates were Gram-negative, lacked argi- soft rot similar to those observed after inoculation with nine dihydrolase, did not produce fluorescent pigment on the reference strains (B. cepacia and B. gladioli). The tis- King B medium, grew aerobically and were able to utilize sues around the inoculation points became yellow or pale arginine, lactose, cellobiose, sorbitol and sucrose. On YDC brown and sunken. These symptoms were visible within 4 medium the colonies were brownish-cream, non-mucoid days post inoculation and progressed as time went by. The and produced a diffusible brownish pigment. Further- disease index differed among studied isolates (Table 4). No more, all isolates were oxidase positive, were able to grow symptoms were observed in any of the controls. at pH 4.0 and 8.0 but not at pH 9.0 and caused distinct According to Lopez et al. (2004) and Alvarez (2004) the necrotic lesions on the leaves of tobacco plants. The same accurate and reliable diagnosis of plant pathogenic bac- biochemical profiles were shown by the reference strain teria requires the use of integrated approaches based on B. gladioli pv. alliicola LMG 6979 (Table 5). On CB and the use of multiple techniques. Accordingly, in the present PCAT media all isolates grew like the reference isolates. case, biochemical and molecular methods were used for The colonies were white, had a flat margins and were ca. the identification of the bacterial isolates under study. 1 mm in diameter. After two-day growth on NA medium, colonies were The results of biochemical tests of all isolates were in round, small, circular and creamy. B. cepacia LMG 6979 agreement with those described for B. gladioli (Schaad, produced a yellowish pigment, probably toxoflavin, a 2001), an identification that was confirmed by PCR us- ing B. gladioli-specific primers (CMG-16-1/G-16-2 and Table 4. Pathogenicity index of bacterial isolates obtained in CMG-23-1/G-23-2) which amplified the expected prod- pathogenicity tests on onion bulbs. ucts of 468 and 388 bp (Fig. 1 and 2). Furthermore the ERIC-PCR profiles of all isolates were identical to that of Bacterial isolates or species Pathogenicity index (0-3)* the reference strain B. gladioli, as shown in Fig. 3 which Bg259 3.0 illustrates the ERIC-PCR profiles of reference strain and Bg260 2.3 Bg261 3.0 of the three representatives of the isolates under study Bg285 3.0 (Bg259, Bg500 and Bg514). Bg294 1.7 All partial sequences of recA and phaC genes of the Bg295 1.7 Polish isolates were identical to the comparable sequences Bg435 1.8 Bg467 2.5 of strain LMG 6979, deposited in BCCM as Burkholderia Bg469 3.0 gladioli pv. alliicola. By contrast, sequences of gyrB and Bg494 3.0 lepA of isolates Bg435, Bg469, Bg494 were identical to Bg500 3.0 those of LMG 6979, but differed from those of the re- Bg514 3.0 maining Polish isolates, so that the isolates under study B. gladioli pv. alliicola LMG 6979 3.0 B. cepacia LMG 6962 3.0 separated into two groups based on the sequences of these Non inoculated control 0.0 two genes (Table 6). Sequences were deposited in Gen- * 0 = no maceration of onion tissue; 1 = limited tissues maceration; 2 Bank (http://www.ncbi.nlm.nih.gov/) with accession Nos = about 50% maceration; 3 = about 100% maceration. KF857488-KF857535. Journal of Plant Pathology (2015), 97 (1), 37-43 Kowalska et al. 41

Table 5. Phenotypic characteristic of pathogenic bacterial isolates from onion bulbs used in this study and reference strain B. gladioli pv. alliicola LMG 6979.

Studied isolates (Bg259, Bg260, Bg261, Bg285, Bg294, Bg295, Reference strain B. gladioli pv. alliicola LMG Test Bg435, Bg467, Bg469, Bg494, Bg500 and Bg514) 6979 Gram reaction − − KOH reaction − − Colour of colony on NA medium creamy creamy Anaerobic growth − − Tobacco HR + + Oxidase + + Production of fluorescent pigment − − on King B medium Colonie colour on YDC medium brownish-cream brownish-cream Growth on D1M medium − − Growth at pH 4.0 + + Growth at pH 8.0 + + Growth at pH 9.0 − − Utilization of arginine + + Growth at 40ºC + + Arginine dihydrolase − − Utilization of lactose + + Utilization of sucrose + + Utilization of sorbitol + + Utilization of cellobiose + + + positive; − negative.

Table 6. Representation of sequence types of analysed strains.

Strain group Sequence type gyrB lepA phaC recA 1 - LMG 6979, Bg435, Bg469, Bg494 1 1 1 1 2 - Bg259, Bg260, Bg261, Bg285, Bg294, 2 2 1 1 Bg295, Bg467, Bg500, Bg514

Fig. 1. Amplification of the 388-bp product of the 16S rDNA gene from Burkholderia gladioli using species-specific PCR primers. Lane 1 and 18, 100 bp DNA ladder; lane 2, B. gladi- oli pv. alliicola LMG 6979; lanes 3-15, isolates Bg259, Bg260, Bg261, Bg285, Bg294, Bg295, Bg435, Bg467, Bg469, Bg494, Bg500, Bg514; lane 16 , B. cepacia LMG 6962; lane 17, water control.

Fig. 2. Amplification of the 468-bp product of the 16S rDNA gene from Burkholderia gladioli using species-specific PCR priners. Lane 1 and 18, 100 bp DNA ladder; lane 2, B. gladi- Fig. 3. ERIC-PCR. Lane 1 – 100 bp DNA ladder (Thermo Sci- oli pv. alliicola LMG 6979; lanes 3-15, isolates Bg259, Bg260, entific), 10 – Bg514, 11 – B. cepacia LMG 6962, 12 – B. gladi- Bg261, Bg285, Bg294, Bg295, Bg435, Bg467, Bg469, Bg494, oli LMG 6979, 15 – Bg259, 19 – Bg500, 20 – B. gladioli LMG Bg500, Bg514; lane 16 , B. cepacia LMG 6962; lane 17, water 6979, lanes 2, 3, 4, 5, 6, 7, 8, 9, 13, 14, 16, 17, 18 – other isolates control. not studied in this work. 42 Burkholderia gladioli in Poland Journal of Plant Pathology (2015), 97 (1), 37-43

Table 7. Megablast crosschecks with obtained sequences types.

Bacterial strain Sequence Identity E-value Query coverage (%) (%) B. gladioli strain BSR3 phaC_1 99 0.0 99 lepA_1 99 0.0 99 Fig. 4. 16S rRNA alignment with distinction of nucleotide differences shown as shadows in the box. lepA_2 99 0.0 99 recA_1 99 0.0 99 Three representative isolates (Bg259, Bg295 and Bg494) gyrB_1 99 0.0 99 were chosen for 16S rRNA sequence identification. The se- gyrB _2 99 0.0 99 quence (accession No. KF857486) was identical for Bg259 B. glumae strain BGR1 phaC_1 94 0.0 99 and Bg295 but differed from that of Bg494 (accession No. lepA_1 92 0.0 98 KF857487) by two base pairs (Fig. 4). lepA_2 92 0.0 98 Genome sequence of B. gladioli strain BSR3 (GenBank recA_1 95 0.0 98 accession No. CP002599.1) and strain gyrB _1 93 0.0 99 BGR1 (GenBank accession No. CP001503.2) were used for megablast crosschecks with the sequences of the Pol- gyrB _2 93 0.0 99 ish isolates (Table 7). As BLASTn search with megablast algorithm (http://blast.ncbi.nlm.nih.gov) against GenBank Coenye T., Vandamme P., 2003. Diversity and significance of nucleotide collection (nr/nt) database of all sequences ob- Burkholderia species occupying diverse ecological niches. tained support the identification of Polish bacterial isolates Environmental Microbiology 5: 719-729. as B. gladioli pv. alliicola the conclusion is that there is a Cowan S.T., 1974. Cowan and Steel’s Manual for Identification complete agreement in the outcome the morphological, of Medical Bacteria. Cambridge University Press, Cam- physiological, biochemical and molecular diagnostic meth- bridge, UK. ods utilized in the present investigation. Drancourt M., Bollet C., Raoult D., 1997. Stenotrophomonas afri- The isolates under study had a patogenicity index rang- cana sp. nov., an opportunistic human pathogen in Africa. ing from 1.7 to 3.0 (Table 4). However, since the majority of International Journal of Systemic Bacteriology 47: 160-163. them had index 3.0 and caused complete rotting of bulbs, El-Hendawy H.H., 2004. Association of pectolytic fluorescent it can be assumed that these isolates are severe onion pseudomonads with postharvest rots of onion. Phytopatho- pathogens. To our knowledge, the present study provides logia Mediterranea 43: 369-376. the first experimental evidence of the presence of B. gla- Fiori M., Ligios V., Schiaffino A., 2011. Identification and char- dioli as dangerous pathogen of onion in Poland. acterization of Burkholderia isolates obtained from bacterial rot of saffron (Crocus sativus L.) grown in Italy. Phytopatho- logia Mediterranea 50: 450-461. REFERENCES Gijon-Hernandez A., Teliz-Ortiz D., Mejia-Sanchez D., De La Torre-Almaraz R., Cardenas-Soriano E., De Leon C., Mora- Agrios G.N., 2005. Plant Pathology. Elsevier-Academic Press, Aguilera A., 2011. Leaf stripe and stem rot caused by Burk- New York, NY, USA holderia gladioli, a new maize disease in Mexico. Journal of Aljanabi S.M., Martinez I., 1997. Universal and rapid salt ex- Phytopathology 159: 377-381. traction of high quality genomic DNA for PCR-based tech- Gitaitis R., MacDonald G., Torrance R., Hartley R., Sumner niques. Nucleic Acids Research 25: 4692-4693. D.R., Gay J.D., Jahnson W.C., 1998. Bacterial streak and Alvarez A.M., 2004. Integrated approaches for detection of bulb rot of sweet onion: II. Epiphytic survival of Pseudomo- plant pathogenic bacteria and diagnosis of bacterial diseases. nas viridiflava in association with multiple weed hosts. Plant Annual Review of Phytopathology 42: 339-366. Disease 82: 935-938. Bauernfeind A., Schneider I., Jungwirth R., Roller C., 1998a. Gitaitis R., Walcott R., Culpepper S., Sanders H., Zolobowska Molecular procedure for rapid detection of Burkholderia L., Langston D., 2002. Recovery of Pantoea ananatis, casual mallei and Burkholderia pseudomallei. Journal of Clinical Mi- agent of center rot of onion, from weeds and crops in Geor- crobiology 36: 2737-2741. gia , USA. Crop Protection 21: 983-989. Bauernfeind A., Schneider I., Jungwirth R., Roller C., 1998b. Hugh R., Leifson E., 1953. The taxonomic significance of fermen- Discrimination of Burkholderia gladioli from other Burkhold- tative versus oxidative metabolism of carbohydrates by various eria species detectable in cystic fibrosis patiens by PCR. Gram-negative bacteria. Journal of Bacteriology 66: 24-26. Journal of Clinical Microbiology 36: 2748-2751. Jacobs J.L., Fasi A.C., Ramette A., Smith J.J., Hammerschmidt Beriam L.O.S., 2007. Palestra doenças bacterianas em hor- R., Sundin G.W., 2008. Identification and onion pathogenic- taliças. Biologico 69: 81-84. ity of Burkholderia cepacia complex isolates from the onion Burkholder W., 1950. Sour skin, a bacterial rot of onion bulbs. rhizosphere and onion field soil. Applied Environmental Mi- Phytopathology 40: 115-118. crobiology 74: 3121-3129. Journal of Plant Pathology (2015), 97 (1), 37-43 Kowalska et al. 43

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Received February 12, 2014 Accepted August 28, 2014