First report of Serratia marcescens causing yellow wilt disease on sunflower in Russia

In August-September 2011, sunflower (Helianthus annuus) plants with yellow, wilted leaves were found in commercial fields in the Republic of North Osetia-Alania (North Caucasus region of Russia). Approximately 20% of the plants in the fields exhibited yellowing and wilting. Cross sections of stems near the affected leaves revealed a brown discoloration in the phloem.  Isolations were made from the stems of 24 symptom-bearing sunflower plants collected from four different fields. Small tissue pieces (2-4 mm³) from the phloem were excised, surface sterilised, rinsed several times and ground. An aliquot of the slurry was plated onto yeast extract-dextrose-calcium carbonate (YDC) agar. Isolates from sunflower plants were consistent with Serratia marcescens in colony colour, morphology and biochemical properties, as described by Schaad et al. (2001).

To determine the genetic diversity, partial sequences of the 16S rRNA gene and recA gene were amplified using primer pairs 8F/1492R (Stackebrandt & Liesack, 1993) and ErecAF/ErecAR (Young & Park, 2007), respectively. Additionally, the bacterial isolates were tested by multiplex PCR with primers YV1/YV4, specific for S. marcescens, and a79F/R which amplifies only cucurbit yellow vine disease (CYVD)-causing strains (Zhang et al., 2005).  The partial 16S rRNA gene sequences (GenBank Accession Nos. KT741016-KT741022) were 99% identical to S. marcescens strains MG1 (AY498856) and JASM1 (KF528829). The recA gene sequences (KT780423-KT780430) were 98% identical to S. marcescens (type strain ICMP 7617; DQ859889).  PCR reactions using primer pairs YV1/YV4 and a79F/R were positive for all isolates tested.

Eight isolates were grown on YDC agar, suspended in sterile water at a concentration of 10⁸ cells/ml and infiltrated into leaves of tobacco (Nicotiana tabacum cv. ‘Xanthi’), and petioles of cotyledon leaves of three-week-old seedlings of sunflower (H. annuus cv. ‘Flagman’) and zucchini (Cucurbita pepo). Sterile water was applied as a negative control. All the inoculated tobacco leaves showed a hypersensitive reaction after 12 hours. The sunflower and zucchini plants were grown for 20 days in a glasshouse at 25/24°C (day/night). The plants were examined for wilting and a cross section was taken along the stem to observe the phloem. 84% of 65 inoculated sunflower and 30% of 27 zucchini plants developed wilting symptoms and phloem discoloration, while all the control plants remained free of symptoms. 

CYVD caused by S. marcescens (Bruton et al., 2003) was first discovered in 1988 in the USA and is now spread throughout many states. The pathogen is vectored by the insect Anasa tristis. In the Russian Federation, plant diseases caused by bacteria of the genus Serratia have not been reported previously. This is the first report of the pathogen in the country and the first report of a new host plant, sunflower.