Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd) sequences exhibit resistance to PSTVd infection. Resistance seems to be correlated with high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the plant. Thus, although small RNAs produced by infecting viroids [small RNAs of PSTVd (srPSTVds)] do not silence viroid RNAs efficiently to prevent their replication, hpRNA-derived siRNAs (hp-siRNAs) appear to effectively target the mature viroid RNA. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21- and 24-nucleotide siRNAs of both (+)- and (-)-strand polarities along the PSTVd genome. These data suggest that RNAi can be employed to engineer plants for viroid resistance, as has been well established for viruses.

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.

The distribution and natural phytoplasma infection of Cacopsylla picta were investigated during a long-term field survey between 2002 and 2009 in commercial and abandoned apple proliferation-infected orchards throughout Germany, northern Switzerland, and eastern France. Comparable population dynamics were described for the different sites whereas considerable variations in the absolute population densities were observed among the years. Individual polymerase chain reaction (PCR) testing revealed, for each year, a rather stable natural infection rate with ?Candidatus Phytoplasma mali? of ?10% for overwintered adults of C. picta. Both genders were equally highly infected although more females were caught. The overall male/female ratio was 1:1.5. No direct correlation was found between the infection status of the orchard and the infection rate of overwintered C. picta. No influence of agricultural practices was seen. However, a relationship between the incidence of the disease and the vector population density became evident on a regional scale. Successful transmission of ?Ca. P. mali? occurred each year with overwintered individuals as well as with new adults. The transmission efficiency varied among the years within 8 to 45% for overwintered adults and 2 to 20% for individuals of the new generation. The load of single C. picta with ?Ca. P. mali? was determined by quantitative real-time PCR. High phytoplasma titers were measured in overwintered adults already at their first appearance in the orchards after remigration from their overwintering hosts. Thus, the data indicate the transmission of the disease on a regional scale by remigrant adults of C. picta and at a local scale within the same season by emigrant adults which developed on infected plants.

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

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