Genome Analysis of Erwinia amylovora Strains Responsible for a Fire Blight Outbreak in Korea.

Erwinia amylovora is a plant-pathogenic bacterium that causes fire blight disease in Rosaceae plants. Since fire blight is highly contagious and results in serious losses once introduced, it is regulated as a quarantine disease. Recently, for the first time in East Asia, fire blight emerged in Korea with strains of E. amylovora being isolated from lesions of infected trees. Five of those strains were selected and subjected to whole-genome shotgun sequencing. Each strain had two circular replicons, a 3.8-Mb chromosome and a 28-kb plasmid. The genome sequences were compared with those of other E. amylovora strains isolated from different hosts or geographical regions. Genome synteny was analyzed and sequence variations including nucleotide substitutions, inversions, insertions, and deletions were detected. Analysis of the population genomic structure revealed that the five strains form a distinct structural group. Phylogenomic analysis was performed to infer the evolutionary relationships among E. amylovora strains, which indicated that the Korean isolates, all descended from a common ancestor, are closely related to a lineage of North American strains. These results provide useful information for understanding the genomic dynamics of E. amylovora strains including those in Korea, developing genetic markers for surveillance of the pathogen or diagnosis of the disease, and eventually developing measures to eradicate it.

A loop-mediated isothermal amplification (LAMP) assay for rapid identification of Ceratitis capitata and related species.

True fruit flies (Tephritidae) are among the most destructive agricultural pests in the world, attacking a wide range of fruits and vegetables. The Mediterranean fruit fly Ceratitis capitata is a highly polyphagous species but, being widely established in the Mediterranean region, is not considered as a EU quarantine pest. Hence, it is important to discriminate Ceratitis capitata from non-EU tephritid species, present in imported fruit and vegetables, as non-EU species have a quarantine status. However, morphological identification of tephritid larvae, the most frequently intercepted stage in non-EU produce, is difficult and an easy-to-use molecular diagnostic tool would be helpful for rapid species identification. Therefore, a loop-mediated isothermal amplification (LAMP) method was developed for C. capitata and non-EU tephritids Ceratitis cosyra group1 and Ceratitis species from the FARQ complex, C. fasciventris, C. anonae, C. rosa and C. quilicii. LAMP assays were run with DNA from ILVO collected specimens and DNA samples collected during previous research surveys. LAMP primers were species-specific, with LAMP amplification occurring within 45 minutes for the targeted species. In addition, LAMP assays were successful for all C. capitata life stages or a limited amount of tissue. To conclude, the LAMP assays developed in this study were able to distinguish C. capitata from non-EU Tephritidae species and could be a useful tool for the rapid identification of C. capitata.

Xylella fastidiosa: Host Range and Advance in Molecular Identification Techniques.

In the never ending struggle against plant pathogenic bacteria, a major goal is the early identification and classification of infecting microorganisms. Xylella fastidiosa, a Gram-negative bacterium belonging to the family Xanthmonadaceae, is no exception as this pathogen showed a broad range of vectors and host plants, many of which may carry the pathogen for a long time without showing any symptom. Till the last years, most of the diseases caused by X. fastidiosa have been reported from North and South America, but recently a widespread infection of olive quick decline syndrome caused by this fastidious pathogen appeared in Apulia (south-eastern Italy), and several cases of X. fastidiosa infection have been reported in other European Countries. At least five different subspecies of X. fastidiosa have been reported and classified: fastidiosa, multiplex, pauca, sandyi, and tashke. A sixth subspecies (morus) has been recently proposed. Therefore, it is vital to develop fast and reliable methods that allow the pathogen detection during the very early stages of infection, in order to prevent further spreading of this dangerous bacterium. To this purpose, the classical immunological methods such as ELISA and immunofluorescence are not always sensitive enough. However, PCR-based methods exploiting specific primers for the amplification of target regions of genomic DNA have been developed and are becoming a powerful tool for the detection and identification of many species of bacteria. The aim of this review is to illustrate the application of the most commonly used PCR approaches to X. fastidiosa study, ranging from classical PCR, to several PCR-based detection methods: random amplified polymorphic DNA (RAPD), quantitative real-time PCR (qRT-PCR), nested-PCR (N-PCR), immunocapture PCR (IC-PCR), short sequence repeats (SSRs, also called VNTR), single nucleotide polymorphisms (SNPs) and multilocus sequence typing (MLST). Amplification and sequence analysis of specific targets is also mentioned. The fast progresses achieved during the last years in the DNA-based classification of this pathogen are described and discussed and specific primers designed for the different methods are listed, in order to provide a concise and useful tool to all the researchers working in the field.

Effects of Long-Wave UV Light on Monilinia Growth and Identification of Species.

The effect of long-wave UV/dark period on mycelial growth of 46 isolates of Monilinia sp. collected in Spain and 16 isolates collected from other parts of the world was investigated. Typical isolates of M. laxa, M. fructigena, and M. fructicola were grown in the dark and identified by morphological characteristics. Long-wave UV/dark conditions reduced the growth rates of M. laxa, M. fructigena, and M. fructicola on potato dextrose agar. All isolates of M. fructigena grew more slowly than those of M. fructicola. Typical and atypical isolates of M. fructigena and M. fructicola were placed in their respective species based on long-wave UV/dark growth rate data. M. laxa isolates were readily distinguished by the short distance from their conidium to the first germ tube branch. The involvement of different photoreceptors in photoresponses by M. fructicola and M. fructigena is discussed. Differences in mycelial growth under long-wave UV may be a useful tool to identify Monilinia spp.