Since 2000, a serious epidemic of apple proliferation (AP) reappeared in southwestern Germany. Molecular analyses revealed that the AP phytoplasma is associated with this disease. Since no curative treatments or resistant cultivars exist, the only means to reduce spread of the disease is the control of the insect vector. Recently, Frisinghelli et al. (1) identified Cacopsylla costalis as a vector of AP phytoplasma in northern Italy. Following this result, transmission trials with C. picta (synonym C. costalis) were conducted in southwestern Germany at Neustadt (Rheinland-Pfalz) and Dossenheim (Baden-Württemberg) since 2001. Overwintering psyllids were captured from March to May in different orchards. Groups of 5 to 30 C. picta were caged for 2 to 4 weeks on apple seedlings or healthy micropropagated plants. Leaf midribs of test plants were sampled 2 to 3 months after inoculation feeding and tested by polymerase chain reaction (PCR) for AP phytoplasma with specific primers AP5/AP4 (2). In 2001, 1 of 10 test plants, and in 2002, 7 of 40 test plants became AP infected. In 2002, one to four C. picta specimens fed on plants which became infected were tested AP phytoplasma positive by PCR while all psyllids recollected from PCR-negative plants were tested negative. Transmission of the AP phytoplasma was successful at both sites. To our knowledge, this is the first report of C. picta as a vector of the AP phytoplasma in Germany. References: (1) C. Frisinghelli et al. J. Phytopathol. 148:425, 2000. (2) W. Jarausch et al. Appl. Environ. Microbiol. 60:2916, 1994.
A quantitative PCR (qPCR) assay was established for a sensitive and specific quantification of apple proliferation (AP) phytoplasmas in plants and in insect vectors. Different AP phytoplasma-specific primer pairs previously selected in a non-ribosomal DNA fragment of AP phytoplasma were tested. Among these, primer pair AP3/AP4 has been chosen for the qPCR assay because it amplifies a small sized 162 bp fragment of AP phytoplasma and produces no artefact bands. Thus, with these primers the SYBR™ Green technology could be used to monitor the amplification of the PCR products in real-time. The absolute quantification of the phytoplasmas in the samples was done by using the standard curve quantification method. The plasmid pUCI196 containing the chromosomal fragment of AP phytoplasmas from which the specific primers were derived was used as standard. Serial dilutions of the plasmid were done in total DNA extracts of healthy plants and healthy psyllids, respectively. For insects, total DNA of single individuals was extracted and subjected to PCR. Thus, AP phytoplasmas could be quantified in single individuals. For plant material, quantification of AP phytoplasmas was done with reference to a defined fresh weight of the material prior to DNA extraction. The inter-assay and intra-assay reproducibility of the method was analysed by comparing the Ct-values for given samples. The reproducibility was high both with plant and insect samples. Great differences in phytoplasma load could be found in different insect vector individuals whereas the analysed plant material was more homogenously infected. The established method is now suitable for the study of the infectivity of the insect vectors as well as for the evaluation of the resistance in plant material
Phytoplasmas are plant-pathogenic, phloem-colonizing, cell wall-less microorganisms that are primarily dependent on insect transmission for their spread and survival. The life cycle of phytoplasmas involves replication in insects and host plants. Until recently, phytoplasmas have resisted all attempts at cultivation in cell-free media, making these pathogens poorly characterized on a physiological and biochemical basis. However, host-pathogen relationships can be studied by investigating immunodominant membrane proteins (IDPs), which are located on the exterior surfaces of phytoplasma cells and are the most abundant proteins of the cell membrane. These membrane proteins come in direct contact with both insect and plant hosts and are thought to play a crucial role in phytoplasma spread both within the plant and by insect vectors. Therefore, there is great interest in studying this class of proteins. We summarize and discuss important investigations about these membrane proteins, which have already provided a better understanding of the host-phytoplasma relationship.
In the post-genomic era, increasingly sophisticated genetic tools are being developed with the long-term goal of understanding how the coordinated activity of genes gives rise to a complex organism. With the advent of the next generation sequencing associated with effective computational approaches, wide variety of plant species have been fully sequenced giving a wealth of data sequence information on structure and organization of plant genomes. Since thousands of gene sequences are already known, recently developed functional genomics approaches provide powerful tools to analyze plant gene functions through various gene manipulation technologies. Integration of different omics platforms along with gene annotation and computational analysis may elucidate a complete view in a system biology level. Extensive investigations on reverse genetics methodologies were deployed for assigning biological function to a specific gene or gene product. We provide here an updated overview of these high throughout strategies highlighting recent advances in the knowledge of functional genomics in plants.
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