Bacterial wilt, caused by Ralstonia solanacearum, on tomato in Italy

S. Loreti^1*, M. Fiori² , D. De Simone¹, G. Falchi², A.Gallelli¹, A. Schiaffino² and S. Ena³

¹ C.R.A. - Istituto Sperimentale per la Patologia Vegetale, Via C. G. Bertero, 22, 00156 Roma, Italy
² Dipartimento di Protezione delle Piante, Università degli Studi, Via E. De Nicola, 07100 Sassari, Italy
³ERSAT - Centro Zonale di Assemini, Via Turati, 3, 09032 Cagliari, Italy

^*s.loreti@ispave.it

Accepted for publication 19/06/07

Symptoms of wilting, resembling those incited by Ralstonia solanacearum, were observed in February and April 2007 on tomato (Lycopersicon esculentum) cultivars Arawak, Ikram and Cuore di bue, growing in five greenhouses in Southern Sardinia (Italy). At first affected plants showed collapse of the growing apex. Stunted lateral shoots emerged but then wilted and died (Fig. 1a) while adventitious roots appeared on the stems. Cross sections of stems showed brown discoloration (Fig. 1b) of the vascular tissue and oozed a dirty white exudate. In April, when environmental conditions were warmer, wilting of the whole plant occurred rapidly. The percentage of wilted plants ranged from 10% to 70%.

Figure 1: Wilting of tomato plant (left) cross section of stem showing brown discoloration of the vascular tissue (above)

Isolations were performed from ten symptomatic plants and from water used for irrigation. Fluidal colonies, which were either entirely white or white with a red centre, were isolated from all samples on SMSA (Elphinstone et al., 1994) and TZCA (Kelman, 1954) media. For pathogenicity tests, six isolates were each inoculated on 9 plants of tomato, eggplant and tobacco. The assay was repeated twice. The stem was wounded with a sterile syringe and a drop of the bacterial suspensions (10⁶-10⁷CFU/ml) were placed on the wound. Control plants were inoculated with sterile distilled water (SDW). Typical symptoms appeared on inoculated tomato and eggplants within one week (Fig. 2); after two weeks all plants had wilted and died. No symptoms were observed on inoculated tobacco and control plants.

Figure 2: Wilting of tomato (left) and eggplant plants (right) experimentally infected with isolate ISPaVe 1322

All isolates and re-isolates were positive for oxidase, catalase, poly-b-hydroxybutyrate and nitrate reduction, oxidatively metabolized glucose and produced alkali from citrate. They did not induce a hypersensitive reaction on tobacco leaves, did not grow at 40°C and did not produce fluorescent pigment on KB, nor levan on NSA nor alkali from arginine. Furthermore they did not hydrolyse aesculin, gelatin or starch. Biolog™ analysis identified the isolates as Ralstonia solanacearum [similarity index match 0.55; probability 99%]. A positive reaction was obtained in IFAS.  PCR, according to Seal et al. (1993), gave the expected band of 288 bp (Fig. 3). The digestion of the amplification products with Ava II gave the same restriction pattern as the R. solanacearum type strain NCPPB 325.

Figure 3: Amplification of 288 bp band of Ralstonia solanacearum from vegetal tissues and water by using OLI-1 and Y-2 primers.1 and 2, type strain NCPPB 325; 3, negative control (Pseudomonas corrugata IPV-SS 98a); 4 and 5, bacterial isolates DPP-RS1 and DPP-RS2; 6 and 7, vegetal tissues of naturally infected tomato plants; 8, vegetal tissues of experimentally infected tomato plants; 9, water used for irrigation; 10, negative control (SDW ). M = 1 kb plus molecular size marker (Invitrogen).

On the basis of the results obtained, the bacteria isolated from tomato, in Sardinia, can be identified as Ralstonia solanacearum (Smith) Yabuuchi et al. This is the first report of the presence of R. solanacearum on tomato in Italy.

References

Elphinstone J, Hennessy J, Wilson J, Stead DE, 1996. Sensitivity of different methods for the detection of Pseudomonas solanacearum (Smith) Smith in potato tuber extracts. EPPO Bulletin 26, 663-678.

Kelman A, 1954. The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44, 693-695.

Seal SE, Jackson LA, Young JPW and Daniels MJ, 1993. Differentiation of Pseudomonas solanacearum, P. syzygii, P. picketti and the blood disease bacterium by partial 16S rRNA sequencing: constructing of oligonucleotides primers for sensitive detection by polymerase chain reaction. Journal of General Microbiology 139, 1587-1594.