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Laboratory of Plant Protection and Biotechnology Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk 24 Kladki Street, 80-822 Gdansk Phone: +48 58 523 6360, Fax +48 58 5236426 Blackleg and soft rot threats, prevention and method of identification Pectobacterium atrosepticum (Erwinia atroseptica) Pectobacterium carotovorum subsp. carotovorum (Erwinia carotovora) Dickeya dianthicola, Dickeya solani (Erwinia chrysanthemi) Prepared by: Professor Ewa Lojkowska, MSc Marta Potrykus, Dr. Wojciech Sledz, Dr. Robert Czajkowski, MSc Agata Motyka, Sabina Zoledowska. LPPB, IFB UG and MUG This work was supported by grants: • • NCN 7322/B/P01/2011/40 Pol-Nor/202448/28/2013 Blackleg and soft rot disease are caused by pectinolytic bacteria Among pathogenic bacteria responsible for losses in potato yield, the pectinolytic species are of the highest concern. They used to belong to the genus Erwinia, but after application of molecular diagnostic methods, they were reclassified to the genera Dickeya and Pectobacterium. These pathogens cause blackleg and soft rot on potato while only soft rot on vegetables and ornamental plants. Nowadays Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum, Dickeya dianthicola and Dickeya solani are the species regarded as the causative agents of soft rot and blackleg in Poland. In addition, the threat of Dickeya was the reason for zero tolerance policy, initiated in 2010 in Scottish potato seed plantations. Moreover, introducing the same treatment on the territory of North Africa is under vivid discussion. In Poland, bacteria belonging to the Dickeya genus were firstly identified on potato seed plantation in 2005. This study was performed by the research group of The Laboratory of Plant Protection and Biotechnology IFB UG and MUG. Since 2009 these microbes are being isolated from seed potato fields every year. Their number varies mainly due to differences in the average air temperature and rainfall during summer. Strategy of protection of potato, vegetables and ornamental plants from pectinolytic bacteria is generally based on the use of diagnostics to test seed stocks for the presence of the pathogen in addition to cleaning and disinfection of equipment used during cultivation, transportation or storage. Pectinolytic bacteria threaten plant vegetation process and storage due to: • • • • • • • The capacity to survive in potato seeds. Easiness to spread in the field resulting from enormous potential to generate economic losses. Ability to survive on plant remains, agricultural implements, sacks, containers. Possibility to cause latent infection. No disease symptoms are present until pathogen – favoring conditions occur. No potato variety resistant to bacteria from the Dickeya and Pectobacterium genera is available on the market yet. Lack of effective chemical and/or biological methods to eliminate these bacteria. Limited knowledge concerning the mechanisms steering the virulence of mentioned pathogens. How to recognize disease symptoms caused by pectinolytic bacteria? There are some disease symptoms listed hereafter that may point to the presence of bacteria from the Dickeya and/or Pectobacterium genera. Importantly, they occur in different stages of plant growth: • • • • Before the emergence of plants - no sprouting, in consequence no plant emerges. After the emergence of plants - primary infections may be observed. They can be distinguished by: yellowish, wilting leaves folding alongside the main vein in addition to blackening and rotting of the base of the shoot. Diseased plants are smaller, usually weakly rooted and it is relatively easy to pull them out from the soil. These plants are the source of bacteria on the plantations. During vegetation - secondary infections may be observed whose symptoms are similar to those of primary infections. Moreover, soft rot may be spotted on plants and tubers. The source of infection are diseased plants, bacteria carried by the wind, rain or insects. More severe symptoms occur in humid, heavy soil during summer. During storage - soft rot of tubers (brown to black pigment may often be observed at the margins of decayed tissue). After mechanical disruption of the peel, the whole tuber disintegrates and the decomposed pulp escapes. The spread of the infection is favored by mechanical damage of the tubers during harvest procedures and the coinfection event with potato pathogens responsible for other diseases like potato blight, dry rot or common scab. Prevention Cultivating only healthy, pathogen-free potato seeds. Cleaning and disinfection of machinery, equipment and grading lines. Regular field inspections during the whole vegetation season. Harvest should be performed under good weather conditions - sunny, no rain. If the crop is collected when it is wet, drying procedures are strongly recommend before placing the tubers in storage. Detection and identification of pectinolytic bacteria with molecular methods Efficient detection and identification of bacteria from the Dickeya and Pectobacterium genera is performed with polymerase chain reaction (PCR). Techniques based on PCR are commonly used for detection of bacteria from the Dickeya and Pectobacterium genera in commercially available potato seeds on the territory Fig. 1. Cutting out the stolon of The Netherlands, Scotland and Finland. ends from potato tubers It is possible to commission diagnostic tests for detection of Dickeya and Pectobacterium in the Laboratory of Plant Protection and Biotechnology IFB UG and MUG. We are the only institution in Poland providing such analysis. Firstly, the plant tissue is collected (Fig. 1), homogenized and the genetic material (DNA) is being isolated. Detection and identification of pectinolytic bacteria is done by Multiplex PCR method capable of revealing the presence of Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum and Dickeya spp. (Fig. 2). Furthermore, PCR-RFLP test allows to confirm/exclude the presence of highly virulent Dickeya solani in tested potato seeds. The Multiplex PCR method was registered as the Polish patent in 2012 and subsequently published (Potrykus et al., Ann App Biol in press). To commission diagnostic tests for detection of pectinolytic bacteria in plant material contact Laboratory of Plant Protection and Biotechnology IFB UG and MUG in Gdansk Fig. 2. Agarose electrophoresis. Multiplex PCR. Pcc - Pectobacterium carotovorum subsp. carotovorum, Pca - Pectobacterium atrosepticum, Dsp – Dickeya sp. Laboratory of Plant Protection and Biotechnology IFB UG and MUG 24 Kladki street, 80-822 Gdańsk Phone +48 58 523 6360, Fax +48 58 5236426 [email protected] [email protected]