Journal of Pathology (2016), 98 (1), 91-96 Edizioni ETS Pisa, 2015 91

BACTERIAL STEM ROT OF GLOBE (CYNARA CARDUNCULUS var. SCOLYMUS) CAUSED BY PECTOBACTERIUM CAROTOVORUM subsp. CAROTOVORUM IN

N. Ustun and N. Arslan

Plant Protection Research Institute, 35040, Bornova, Izmir, Turkey

SUMMARY 11th place among major producers, with 32,173 MT produc- tion and ca. 23 million dollar value (www.faostat.fao.org). In the autumn of 2012 unusual symptoms on globe ar- In Turkey, the cultivation of the crop is concentrated tichoke (Cynara cardunculus var. scolymus) cultivar A 106 mainly in the Aegean and Eastern Marmara Regions, were observed in commercial fields in Aydın province with main producing provinces Izmir, Aydın and Muğla. (Şahnalı) in the Aegean Region of Turkey. Diseased These provinces provide more than half of total country showed stem rot and pith necrosis symptoms at the stem production (Paylan et al., 2013). For many years cvs Sakız base and collapsed soon after first symptoms appeared. and Bayrampaşa have been the most common traditional The disease prevalence was estimated to approach 10-15% cultivars propagated asexually (Temirkaynak et al., 2008). in different fields, leading to removal and destruction of In recent years the production of new, seed-propagated, many plants by the growers. Representative strains iso- cultivars having advantages in productivity, quality and lated from infected plants were identified on the basis of profitability (Temirkaynak et al., 2009; Mauro et al., 2011) morphological, biochemical, sequencing and pathogenic- has steadily increased. Although production by seeds has ity tests as Pectobacterium carotovorum. The identification a disadvantage of lateness, these cultivars can be inserted was further confirmed by sequencing the pel housekeep- in specific cropping systems, where traditional early geno- ing gene of one representative isolate (GenBank accession types are integrated with new late seed-propagated cul- No. KT828557), which was found to have 100% nucleotide tivars, thus extending the temporal availability of heads identity with strain HNXDT002 (GenBank accession No. (Mauro et al., 2011). JF721960.1) of P. carotovorum subsp. carotovorum, isolated Artichoke suffers from abiotic as well as different kind previously from artichoke plants with stem rot symptoms of biotic disorders. Apart from viruses and fungi, bacteria in China. To our knowledge this is the first report on bac- can damage artichoke seriously. Bacterial bract spot of ar- terial stem rot of artichoke in Turkey. tichoke, caused by Xanthomonas cynarae sp. nov. (Trébaol et al., 2000) is a bacterial disease reducing the marketabil- Key words: identification, biochemical tests, PCR, pel ity of the harvest and was observed in France since 1954 gene, sequencing, pathogenicity (Trébaol et al., 2000). Stunted plant growth, plant collapse and soft rot of crown tissue are the main symptoms of bacterial crown rot of artichoke commonly observed in many production areas in the world and caused by Dick- eya chrysanthemi (Smith et al., 2008). The bacterial soft rot symptoms on artichoke caused by Pectobacteriım carotovo- rum (mainly subsp. carotovorum), another bacterial species INTRODUCTION belonging to soft rot erwinias group, may cause soft rot and wilting in the field and can be a problem in storage The globe artichoke (Cynara cardunculus L.) is a peren- and distribution if optimum temperature is not maintained nial plant naturally occurring in the , (Gallelli et al., 2009; Sulow and Cantwell, 2013). P. caro- used as vegetable since the ancient Greek-Roman period. tovorum subsp. carotovorum induced different symptoms A variant, Cynara cardunculus subsp. scolymus (L.) Hayek on globe artichoke in 2008-2010 in China. The disease is now cultivated mainly in the countries bordering the observed in China was named artichoke bacterial stem rot Mediterranean basin. According to FAO statistics for 2012, and was characterized by wilting and necrosis of the outer- is the biggest producer of artichoke in the world, most leaves and dark brown discoloration of the vascular followed by countries such as Italy, , Peru, , tissue and pith of the stem base (Gao et al., 2011). China and . In the world ranking Turkey takes the In Turkey, bacterial wet rot disease (with local name “enginar ya çürüklüğü”) caused by P. carotovorum has Corresponding author: N. Ustun ş Fax: +90.232.3741653 been observed for many years in many production areas E-mail: [email protected] (Anonymous, 2008). Commonly the disease, affecting 92 Bacterial stem rot of globe artichoke in Turkey Journal of Plant Pathology (2016), 98 (1), 91-96 mainly budding flower heads, starts with soft rot of the prepared from each isolate. Bacterial cells were lysed by middle bracts of the flower head, under conditions of rain, heating for 15 minutes at 95°C and subsequently cooled on hail and lower temperatures, in the flower head forma- ice for 10 min and then centrifuged for 3 minute at 7,000 tion stage. In the autumn of 2012 unusual symptoms on rpm. The supernatant was stored at −20°C and was used artichoke cultivar A 106 (Hybrid), known to have a low as target DNA in the PCR. vernalization need, increased productivity (approximately The PCR test was performed according to Darrasse et 30 heads per plant) and high quality of heads, were ob- al. (1994) using Y1/Y2 primers and Thermo Scientific PCR served in commercial fields (approximately 10 ha) in Aydın Master Mix (2×) including 4 mM MgCl2; 0.4 mM of each province (Şahnalı) in Aegean Region of Turkey. The dis- dNTPs 0.05 U/µl Taq DNA polymerase. The one reaction ease prevalence was estimated to approach 5-10%. Dis- mix (50 µl) contained 25 µl PCR Master Mix (2×), 1 µl of eased plants showed stem rot and pith necrosis symptoms each primer (10 mM), 21 µl nuclease-free water and 2 µl at the stem base and collapsed soon after first symptoms template DNA. The mixture was subjected to following appeared. The disease led to removal and destruction of conditions: 1 cycle of 5 min at 94o, 35 cycles of the 30 s at many plants by the growers. 94oC, 30 s at 65oC and 45 s at 72oC and finally 1 cycle of The aim of this study was to identify the causal agent 5 min at 72oC. Amplified DNA was detected by electro- of the stem rot disease on artichoke by using conventional phoresis in a 1.5% agarose gel in 0.5 XTBE buffer and and molecular methods. stained with 5 mg ml−1 of ethidium bromide. The expected fragment length of amplicons was 434 bp.

MATERIALS AND METHODS Pathogenicity. Artichoke plants (cultivar A 106) grown in pots in growth room were artificially inoculated at the Bacterial strains. In biochemical, PCR and pathoge- 5-7 leaf stage by injecting 0.5 ml of a bacterial suspension nicity tests, four representative Turkish isolates, obtained in sterile water adjusted to108 CFU ml−1 into stem at leaf from artichoke plants in 2012, were compared with the bases. Three plants per isolate were used. Control plants type strain for P. carotovorum subsp. carotovorum strain were inoculated with sterile distilled water. Inoculated (CFBP (Collection Française des bactéries phytopatho- plants were incubated at 25 ± 1oC, 5000× light for 12 hour gens) 2046=NCPPB 312), isolated from Solanum tuberosum photoperiod and 80-100% RH for 5-7 days. Re-isolations in 1952 in Denmark and a reference strain for P. carotovo- were performed from artificially inoculated symptomatic rum subsp. atrosepticum (CFBP 1546), isolated from Lyco- plants. Re-isolated bacteria were identified by biochemical persicon esculentum in 1973 in France. and PCR tests.

Isolation. Small (diameter 9-10 mm) stem pieces cut from the border between healthy and rotted tissue were RESULTS surface disinfected in 70% ethanol, rinsed twice in sterile water and macerated in 2 ml 50 mM potassium phosphate Symptoms. Plants with symptoms showed yellowing buffer (Na2HPO4 4.26 g; KH2PO4 2.72 g, distillate water and wilting of the outermost leaves (Fig. 1) which pro- to 1 l; pH 7.0) using a disinfected sterile pestle and mortar. gressed inwards, brown discoloration and necrosis of the Aliquots of 0.1 ml of tenfold serial dilutions in PB (10−3, vascular tissue and pith of the stem base (Fig. 2), result- 10−4) were plated onto nutrient sucrose agar (NSA = Ox- ing in hollowing of the stem, which led to plant collapse oid nutrient agar supplemented with 5% w/v sucrose) and and death. Sometimes new lateral shoots around the dead King’s Medium B (King et al., 1954). Plates were incubated stalks emerged but they commonly were not able to pro- for 4 days at 28°C. Whitish colonies, consistently isolated duce flower heads. Such shoots were unhealthy and un- from diseased tissue, were transferred to nutrient agar marketable with stunted growth and leaves lobed more (NA, Oxoid) to obtain pure cultures that were used to deeply than usual. Stunting and excessive deeply lobbing characterize the isolated bacteria. of leaves, resembling virus disease (like Cucumber mosaic virus in tomato which increases the lobbing in leaves as Biochemical and physiological tests. Gram stain, oxi- it was stated in Zuo et al., 2011), leading in some cases to dase, potato soft rot, arginine dehydrolase, starch hydroly- misidentification of the disease symptoms. o sis, H2S production, gelatin hydrolysis, growth at 37 C, tol- erance to 5% and 7% NaCl, indole production, reducing Isolation. Colonies with grey-white, translucent, glossy compounds from sucrose and utilization carbonhydrates and round appearance were consistently isolated from the were performed as described by Lelliott and Stead (1987) edge of diseased and healthy parts of the plants on NSA and Schaad et al. (2001). (1.0-2.0 mm in diameter after 2 days of growth). On King’s B medium (King et al., 1954) colonies were non fluores- PCR test. For DNA isolation, a suspension of approxi- cent, whitish and round. mately 106 cells per ml in molecular-grade sterile water was Journal of Plant Pathology (2016), 98 (1), 91-96 Ustun and Arslan 93

Fig. 1. Wilting and rotting symptoms of the outermost leaves of artichoke plants.

Fig. 2. Brown discoloration and necrosis of the vascular tissue and pith of the infected artichoke plants.

Biochemical tests. Results of biochemical and physi- the type strain of P. carotovorum CFBP 2046 and the refer- ological tests are shown in Table 1. ence strain for P. atrosepticum CFBP 1546. No amplified Isolated strains were pectolytic, facultative anaerobic, fragment was observed with sterile water (Fig. 3). nonfluorescent and able to hydrolyse esculin and gelatine, to produce H2S, to grow at 37°C and 5% NaCl, to assimi- Pathogenicity tests. In inoculated artichoke plants late mannitol, melibiose, m-inositol, D+mannose, lactose, (cultivar A 106) symptoms resembling those observed on citrate, raffinose and ethanol. They gave negative reaction the naturally infected plans were observed 4-5 days post for Gram staining, oxidase, levan production, arginine di- inoculation (Fig. 4). hydrolase, starch hydrolysis, indole production, sensitivity The symptoms started with brown-black discoloration to erythromycin, reducing compounds from sucrose, tol- and soft rot of stem tissue at the inoculated sites. Rotting erance to 7% NaCl and production of acid from sucrose, progressed toward the petiole and leaf blade. Midrib of sorbitol, maltose, adonitol and dulcitol. The artichoke iso- the leaves and neighboring tissue became black and had lates showed the same biochemical characters as the type a water-soaked appearance. In longitudinal section of the strain of P. carotovorum subsp. carotovorum, CFBP 2046. affected stem apart from discoloration also disintegration These tests also excluded the related pathogens P. betavas- of the tissue and hollowing and necrosis of the pith were culorum, P.c. subsp. odoriferum, P. wasabiae and Dickeya observed. Eventually plant collapsed and died. No symp- spp. toms were observed on control plants inoculated with ster- ile water. The bacteria re-isolated from inoculated plants PCR tests. An amplified fragment of 434 bp was ob- were re-identified on the basis of biochemical and PCR tained from all representative artichoke isolates as well as tests as P. carotovorum subsp. carotovorum. 94 Bacterial stem rot of globe artichoke in Turkey Journal of Plant Pathology (2016), 98 (1), 91-96

Table 1. Results of biochemical and physiological tests.

CFBP 2046 Turkish CFBP1546 Test P. c. subsp. artichoke P. atrosepticum carotovorum isolates (4)

Gram reaction − − − Levan formation on NSA − − − Oxidase − − − Soft rot on potato slices + + + Arginine dihydrolase − − − Oxidative/Fermentative Fermentative Fermentative Fermentative metabolism of glucose Esculin hydrolysis + + + Starch hydrolysis − − − Gelatin hydrolysis + + + H2S production + + + Indol production − − − Sensitivity to − − − erythromycin Growth at 37 o C + − + Fig. 3. Agarose gel electrophoresis of PCR-amplified DNA of Reducing compounds − + − from sucrose several artichoke isolates and reference strains using pel gene Tolerance to 5%NaCl + + + primers. L: DNA ladder; Lane 1, Pectobacterium carotovorum Tolerance to 7% NaCl − − − subsp. carotovorum, CFBP 2046; lane 2, Pectobacterium atro- Acid from: septicum CFBP 1546; lanes 3-6, representative Turkish arti- Sucrose − − − choke strains; W, sterile water. Sorbitol − − − Mannitol + − + Melibiose + + + The identification was further confirmed by sequencing Maltose − + − the pel gene of one of the representative strains (GenBank m−inositol + + + accession No. KT828557) which was found to have a 100% Adonitol − − − nucleotide identity with strain HNXDT002 (GenBank ac- D+ mannose + + + Lactose + + + cession No. JF721960.1) of a P. carotovorum subsp. caroto- Citrate + + + vorum strain isolated from artichoke showing the similar Raffinose + + + stem rot symptoms in China (Gao et al., 2011). Dulcitol − − − P. carotovorum is an ubiquitous bacterium, able to sur- Ethanol + − + vive in the rhizosphere (Butler, 1980), plant debris (Zielkie, CFBP (Collection Française des bactéries phytopathogens), + = posi- 1989) and water (Mc Carte et al., 1985). This pectolytic tive reaction; − = negative reaction bacterium is known for its ability to cause soft rot disease on a wide range of plants, mostly vegetables and ornamen- Sequencing. Sequences of pel gene of one of the rep- tals, as it produces many plant cell degrading enzymes to resentative Turkish strains (GenBank accession No. macerate different plant organs and tissues, resulting in se- KT828557, protein_id=ALG64173.1) had 100% nucleo- rious damages on different hosts in field and storage stage tide identity with strain HNXDT002 (GenBank acces- worldwide (Kettani-Halabi et al., 2013; Czajkowski et al., sion No. JF721960.1) of P. carotovorum subsp. carotovo- 2015). As in many countries in the world, P. carotovorum rum isolated from artichoke stem rot in China (Gao et al., subsp. carotovorum is one of the pathogens responsible for 2011). Also there was 96% similarity with GenBank ac- potato soft rot in Turkey (Benlioğlu, 1991; Anonymous, cession CP003776.1 and 94% with GenBank accession 2008). It causes also stem rot and pith necrosis on green- CP001657.1, which are strains of P. carotovorum subsp. house tomatoes in the Aegean and the Mediterranean part carotovorum available in GenBank. of the country (Aysan, 2001; Üstün and Saygılı, 2001). An outbreak of P. carotovorum subsp. carotovorum on Dief- fenbachia (Dieffenbachia amoena) in the Eastern Mediter- DISCUSSION ranean Region of Turkey was reported since early 2000 (Çetinkaya-Yıldız et al., 2004). P.c. subsp. carotovorum in- In our study, the causal agent of a bacterial soft rot dis- duced also bacterial leaf, peduncle and bulb soft rot on tu- ease observed on artichoke plants (cultivar A 106 Hybrid) lip in seed-bulb-producing area located in Konya province occurring in commercial fields in Aydın province (Şahnalı) of Turkey (Boyraz et al., 2006). of Aegean Region of Turkey in 2012 was diagnosed. Iso- On artichoke, bacterial wet rot disease has been ob- lates obtained from infected plants were identified on served for many years in some production areas in Tur- the basis of morphological, biochemical, sequencing and key (Anonymous, 2008). This wet rot disease starts with pathogenicity tests as P. carotovorum subsp. carotovorum. blackening and rotting of bracts of budding flower-heads. Journal of Plant Pathology (2016), 98 (1), 91-96 Ustun and Arslan 95

Fig. 4. Symptoms on artificially inoculated plants. Black discoloration and soft rot of stem tissue at the inoculated sites, rotting progressed toward petiole and leaf blade midrib.

Diseased flower-heads develops slowly resulting in little plants in severely infected fields, causing yield losses. The heads formation. In wet conditions the disease may prog- affected area was nearly 10 ha and disease prevalence was ress to the leaves and other parts of the plants which may estimated to be 10%. collapse (Anonymous, 2008). These symptoms are dif- To our knowledge this is the first report on bacterial ferent from the stem rot disease described in this article. stem rot of artichoke in Turkey. The main difference is in the starting point of the diseases caused by the same pathogen. Bacterial wet rot affects pri- marily the bracts of the budding flower-heads and rot- REFERENCES ting may expand to the other part of the plants only in prolonged wet conditions (Anonymous, 2008). However, Anonymous, 2008. Technical Instructions for Plant Protection. symptoms of the disease here named ‘stem rot’ include Vol. 3. Ankara, Turkey. yellowing and wilting of outermost leaves, brown discol- Aysan Y., 2001. Bacterial stem necrosis of tomato in the green- oration and necrosis of the vascular tissue and pith at the houses in the Eastern Mediterranean Region of Turkey. Pro- stem base, stunted shoots with more deeply lobed leaves ceedings of the 11th Congress of the Mediterranean Phyto- and reduced ability to produce budding flower-heads and pathological Union and 3rd Congress of the Sociedade Portu- guesa de Fitopatologia, Evora, 17-20 September 2001: eventually plant collapse and death. 301-303. Stem rot symptoms observed on artichoke grown in Benlioğlu K., 1991. Investigations on distribution rates, identi- Aydın province of Turkey in 2012 were similar to the arti- fication and inoculum sourses of Erwinia spp. in potato pro- choke disease reported in China, characterized by wilting duction areas of Bolu, Nevşehir and Niğde provinces. Plant and brown discoloration of the vascular tissue and pith Health, Faculty of Agriculture, Ege, Izmir, Turkey. of the stem base (Gao et al., 2011). The bacterial isolates Boyraz N., Bastas K.K., Maden S., Yasa A., 2006. Bacterial leaf obtained from stem rot symptoms in our study were identi- and peduncle soft rot caused by Pectobacterium carotovorum fied as P. carotovorum subsp. carotovorum, most similar to on tulips in Konya, Turkey. Phytoparasitica 34: 272-280. those obtained from artichoke in China. The properties Butler L.D., 1980. Erwinia carotovora subsp. carotovora, a com- of these strains have to be fully investigated in order to petative rhizosphere inhabitant of tomatoes and cucumbers, understand if they are different from other P. carotovorum Ph.D. Thesis. University of Arizona, USA. strains causing damages on artichoke in Turkey and other Czajkowski R., Pérombelon M.C.M., Jafra S., Lojkowska E., parts of the world. Potrykus M., van der Wolf J.M., Sledz W., 2015. Detection, An emergence of a new disease on artichoke may re- identification and differentiation of Pectobacterium and sult from introduction of new varieties in the area and Dickeya species causing potato blackleg and tuber soft rot: unusual climatic conditions due to climate changes and a review. Annals of Applied Biology 166: 18-38. new cultural practices considered to pose a challenge to Çetinkaya-Yıldız R., Mirik M., Aysan Y., Küsek M., Şahin F., many pathosystems in recent years. In fact, farmers in 2004. An outbreak of bacterial stem rot of Dieffenbachia amoe- Aydın province argued that the new seed producing arti- na caused by Erwinia carotovora subsp. carotovora in the east- ern Mediterranean Region of Turkey. Plant Disease : 310. choke variety A 106 hybrid produces many budding flower 88 heads (around 30), has a more vigorous growth and more Darrasse A., Priou S., Kotoujansky A., Bertheau Y., 1994. PCR and Restriction Fragment Length Polymorphism of a pel succulent plant tissues, compared to the traditional local gene as a tool to identify Erwinia carotovora in relation to varieties. This vigorous and succulent growth may predis- potato diseases. Applied and Environmental Microbiology pose the plants to stem rot and pith necrosis under certain 60:1437-1443. climatic conditions, similarly to what observed in tomato FAO statistics, 2012. Available at: http://www.faostat.fao.org77. and many other plants (Scarlet et al., 1978; Üstün, 2012). Gallelli A., Galli M., De Simone D., Zaccardelli M., Loreti S., Bacterial stem rot led to destruction of many artichoke 2009. Phenotypic and genetic variability of Pectobacterium 96 Bacterial stem rot of globe artichoke in Turkey Journal of Plant Pathology (2016), 98 (1), 91-96

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Received June 18, 2015 Accepted November 2, 2015