1.6.6
PHYTOTOXIN PRODUCED BY PSEUDOMONAS CICHORII: A PATHOGEN OF LETTUCE BACTERIAL ROT AND ITS PARTIAL CHARACTERIZATION

T SHIRAKAWA1, K OZAKI2 and S HORIUCHI1

1National Research Institute of Vegetables, Ornamental Plants and Tea, Ano, Mie 514-2328, Japan; 2Depertment of Citriculture, National Institute of Fruit Tree Science, Kuchinotsu, Nagasaki 859-2501, Japan

Background and objectives
Bacterial rot of lettuce caused by Pseudomonas cichorii was first reported in the USA in 1956. Since then, the disease has been occurring in many countries of the world. In 1979, Tsuchiya first identified one of the causal agents of the disease in Japan to be P. cichorrii. At present, bacterial rot caused by the pathogen is one of the most serious diseases for summer season lettuce in the highlands, such as Nagano and lwate prefectures. While quite important, mechanisms of pathogenicity have scarcely been studied. Elsewhere, pathogenesis-related substances have been found in many plant-pathogenic bacteria. In the present study, we describe a new phytotoxin-like substance produced by P. cichorii that induces lesions similar to the symptoms on lettuce.

Materials and methods
Bacterial strain P. cichorii YM8705 was mainly used in this study. The strain was isolated from cultivated lettuce at lwate prefecture and identified through the study of bacterial characters and then preserved by freezing at -40C or freeze-drying. Lettuce plants (cv. My-lettuce, Kaneko Seeds Co.) were grown for 2 months (ca 20-25 leaf age) in a greenhouse in 12-cm polyvinyl pots. For phytotoxicity assay, about 6-cm-long leaf midrib pieces were employed.

The strain YM8705 was grown in YPD medium at 28C for 72 h, and then bacterial cells were separated out of the culture. Extraction was done from the bacterial cells with 90% acetone for 2 h at room temperature. The extract obtained was dissolved in 100% methanol and diluted 100 times with distilled water. Lettuce midrib was injected with the extract solution under the epidermis, and incubated at 25C for 48 h in a moist petri dish.

Results and conclusions
A phytopathogenic or pathogenesis-related substance was extracted from the bacterial body of P. cichorii and purified with silica gel chromatography and HPLC using a C-18 column. Not all P. cichorii strains produced the substance. Bacterial species other than P. cichorii did not produce the substance. Brown lesions similar to those caused by the pathogen appeared within 24 h when the substance was injected under lettuce epidermis. The lesion was limited to an area where the substance had been introduced. No yellow halo was observed around the lesion. The substance, as well as P. cichorii, caused lesions on all crops of 54 species tested, including 18 families. Symptom expressions on lettuce induced by both the substance and P. cichorii were inhibited by pre-treatment with cycloheximide and blasticidin-S (protein synthesis inhibitors). The substance was a peptide with molecular weight 2068 Da and designated 'chichorin'.

References
1. Tsuchiya Y, Ohata K, lemura H et al., 1979. Bulletin of the National Institute for Agricultural Science C34, 51-73.