Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative-PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is micro-array technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms.
A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 l of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.
Eukaryotes have established symbiotic relationship with microorganisms, which enables them to accomplish functions that they cannot perform alone. In the German cockroach, Blattella germanica, the obligate endosymbiont Blattabacterium coexists with a rich gut microbiota. The transmission of Blattabacterium is vertical, but little is known about how the gut microbiota colonizes newborn individuals. In this study, we treated B. germanica populations with rifampicin, a broad-spectrum antibiotic, during two generations and analyzed gut bacterial composition and the Blattabacterium load in control and rifampicin-treated populations. Rifampicin exerted a drastic effect on gut microbiota composition, which recovered in the second generation in the case where the antibiotic was not added to the diet. Furthermore, we observed that bacterial species present in the diet, and particularly in the feces, contribute significantly to establishing the gut microbiota. Finally, the Blattabacterium population remained unaffected by the antibiotic treatment of adults during the first generation but was strongly reduced in the second generation, suggesting that this intracellular symbiont is sensitive to rifampicin only during the infection of the mature oocytes, when it is in an extracellular stage.
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