BEHAVIOUR OF BIOLUMINESCENT RALSTONIA SOLANACEARUM CONTAINING THE LUX CDABE OPERON INTOMATOES SUSCEPTIBLE AND RESISTANT TO BACTERIAL WILT
Hikichi Y.1, Nasu Y.2, Toyoda K.2, Suzuki K.2, Horikoshi M.3, Hirooka T.3, Okuno T.1
1
2 Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024, Japan.
3 Research Center, Nippon Nohyaku Co. Ltd., 345 Oyamada, Kawachinagano, Osaka 586, Japan.
Ralstonia solanacearum OE1-1 was transformed with pNP126, which carried the lux CDABE operon derived from Vibrio fischeri and a promoter region derived from P. glumae genome DNA. Transformants were screened for their bioluminescence, plasmid maintenance and pathogenicity in tomato plants. A bioluminescent transformant, R. solanacearum YN5 was selected and used in the experiments.
Tomato plants used were cultivar Oogata-Fukuju for the one susceptible to the disease and cultivars LS-89, BF-Okitsu, Helper-M and Anchor-T for resistant ones which were usually used as the rootstocks of the susceptible scions in Japan. Five week-old tomato plants were inoculated with R. solanacearum YN5 by the root dipping method and were grown in the water culture at 25 C. Bioluminescence derived from R. solanacearum YN5 was observed under a Video-intensified microscopy camera. The collars and the mid of stems of infected Oogata-Fukuju plants emitted strong and weak bioluminescence 3 DAI, respectively. Population of R. solanacearum YN5 in the collar was 2.46 x 107 cfu/g fresh weight, and that in the mid of stem was less than 8 x 102 cfu/g fresh weight, the minimum limit for detection. At 5 DAI, strong bioluminescence was observed in the stem in which the bacterial population was more than 109 cfu/g fresh weight. These results suggested that the degree of bioluminescence derived from R. solanacearum YN5 was positively related with the bacterial population in tomato plants.
Oogata-Fukuju plants inoculated with R. solanacearum YN5 began to wilt 5 DAI and all plants were severely wilted by 15 DAI. No LS-89 plants wilted by 15 DAI. However, BF-Okitsu 101, Helper-M and Anchor-T plants wilted in 58.3, 16.6 and 27.3 % of the plants 15 DAI, respectively.
Bioluminescence in the inoculated Oogata-Fukuju plants was first detected in the roots and the collars 1 DAI and the intensity of bioluminescence became strong in the collars 2-7 DAI. Thereafter, it became weak 8-10 DAI and was not detected 15 DAI. In the mid and the top of stems, bioluminescence was first detected 4 DAI and the intensity became strong 7-8 DAI. Thereafter, the intensity of bioluminescence decreased similarly as in the collars.
In LS-89 plants, bioluminescence was observed only in the roots and collars and the intensity was weaker than that in the roots and collars of Oogata-Fukuju plants. This was the same for the non-wilted plants of BF-Okitsu 101, Helper-M and Anchor-T plants. In the wilted plants of these cultivars, however, bioluminescence was observed similarly as that in the susceptible Oogata-Fukuju plants.
These results directly demonstrated several factors that are important to determine the susceptibility and resistance of tomato to bacterial wilt. The factors are the degree of the multiplication of R. solanacearum in roots and collars after the bacterial invasion from roots, the bacterial spread to the upper stem and bacterial multiplication in the upper stem. Moreover, limitation of the bacterial invasion into the roots was not the sole factor that is related with resistance of LS-89 plants to bacterial wilt because R. solanacearum was found to latently infect in the roots and collars of LS-89 plants.
