ALBERT

Description / Table of Contents: Summary Erwinia carotovora var.atroseptica (Eca) can survive for many years in tissue culture without signs of bacterial infection on the plants or of it being visible on the medium. Because the infection can be transmitted from one subcultivation to the next, there is a need for appropriate tests to detect latent contamination. Latent infection of in vitro plants, glasshouse plants and tubers by Eca was studied using ELISA and two nutrient media tests in trials to determine the levels of detection which could be achieved. Attempts were made to improve the interpretation of the ELISA results by comparing it with other methods of analysis. The plant material originated from in vitro material of known disease status. Sap samples for ELISA were pressed from whole in vitro plants, pieces from the stem bases of glasshouse plants and the heel-end of tubers. ELISA readings were taken at 405 nm using two photometers. The level used for discriminating between negative and positive results was based on calculating $$\bar x + 3s$$ (mean and spread of values respectively). YEB-medium in liquid form and the Luria-Bertani Medium in liquid and solidified (agar) form were the test media (Table 1). Finely cut nodal segments of in vitro plants were placed in liquid media and incubated for five weeks. In vitro leaves which had been pierced were laid on the agar medium. ELISA proved to be inadequate when the results from repeated subcultures were compared (Table 2). The reproducibility of the results varied in sign and value. Readings obtained with negative samples were in better agreement with the disease status of the plants than those from positive ones. Using calculated separation levels gave sharper differentiation in the results than using fixed values. The ELISA results, interpreted on the basis of $$\bar x + 3s$$ , varied considerably when applied to in vitro plants in the glasshouse (Fig. 1). Complete detection of the bacteria was only successful in the first test with in vitro plants and in tuber tests. Detection of latent Eca contamination was unsuccessful using the test nutrient media. Only 1 in 116 replicates gave the clouding expected with the nutrient media. There was strong growth of bacteria after two days in all tests with dilutions of pure cultures. False positives did not occur i.e. tests withErwinia-free clones remained clear. On the basis of these results a clear indication of latent Eca contamination on in vitro plants is not possible using ELISA, since the same clones gave positive or negative results depending on the time of testing. The poor reliability of the test must be due to the levels of the pathogen on in vitro plants (102/g), lower than the detection level of ELISA (103 cells ml−1). The failure of latent bacteria to grow in the test nutrient media may be due to the attachment of the Eca pathogen to the cell wall structure of the in vitro plants. For reliable detection an enzymatic pre-treatment is presumably necessary, which would allow the pathogen to grow actively in the test media. No improvement of detection levels can be expected using ELISA. Glasshouse grown plants give inconsistent results similar to in vitro cultures. The degree of detection achievable with tuber tests renders it impractical, since the danger of recontamination by Eca during tuber multiplication in the glasshouse is too great. However other approaches to detection described in the literature are either uncertain or too expensive, and it appears that there is no alternative to ELISA for the detection of latent Eca contamination. The possible danger of false results can be counteracted to some extent by the combined use of replication and evaluations based on calculated separation values.