Advantages of an integrated approach for diagnosis of quarantine pathogenic bacteria in plant material

Abstract

The accurate and reliable diagnosis of quarantine bacteria and their detection in asymptomatic material requires the use of an integrated approach based on the use of several techniques. This strategy is more expensive and time consuming but it combines conventional, serological and molecular tests in order to get better results. Bacterial isolation is required for having pure cultures necessary for target identification and to perform pathogenicity tests. However, its results can be negative, especially for slow growing pathogens, or when the bacteria in the samples are in the viable but non culturable state. Serological tests, especially indirect immunofluorescence (IIF) and ELISA are very useful for routine testing. Nevertheless, both can have specificity problems due to the poor quality of some available antibodies. The first one has usuallyan acceptable sensitivity and can be employed as a screening test. On the opposite, ELISA sensitivity is, in general, poor and a previous enrichment step is recommended. The use of PCR has improved the sensitivity and specificity of the diagnosis and in many different variants, is a very efficient method for rapid screening of samples. However, there are manycases where the presence of inhibitors gives false negative results, or for which the positive ones by PCR cannot be confirmed with anyother technique. Bioassays have also proved their usefulness, especially for recovering stressed or low populations of bacteria. Theyrequire few weeks for being performed when doing the biological enrichment in planta, but other possibilities such as in vitro growing plants or detached leaves, can also be used. The experience in applying this integrated methodology for diagnosis (isolation, serological tests, PCR and bioassays) has allowed to detect more accurately Erwinia amylovora, Ralstonia solanacearum and Xanthomonas axonopodis pv. citri in plant material.