Flavescence dorée (FD) and Bois noir (BN) are the principal grapevine yellows diseases in Europe caused by distinct phytoplasmas: FD by 16SrV phytoplasmas (FDp), BN by Candidatus Phytoplasma solani. FDp is spread epidemically by the introduced Nearctic Deltocephalinae Scaphoideus titanus and is listed as a quarantine pest in the European Union (Regulation (EU) 2019/2072). Black Alder (Alnus glutinosa) is a common asymptotic host of 16SrV phytoplasmas in Europe and considered the original host of FDp (Malembic-Maher et al. 2020). Palatinate grapevine yellows (PGY) transmitted from alder to grapevine by the Macropsinae Oncopsis alni (Maixner et al. 2000) is not transmissible by S. titanus, unlike isolates transmitted by the autochthonous Deltocephalinae Allygus spp. and the invasive Orientus ishidae (Malembic-Maher et al. 2020). Germany is considered free from FD in grapevine and from its vector. A single case in a nursery in 2014 was eradicated (EPPO 2017). Since S. titanus was detected in 2016 in the neighboring French Region of Alsace, monitoring of FD was carried out in Germany. It was focused on vineyards within a distance of 100 m from stands of alder trees. A geodata-based risk map (Jalke 2020) was used to identify those plots. All symptomatic vines sampled until September 2020 proved to be infected by BN or, occasionally, by PGY. Eight vines with typical symptoms were sampled in vineyards adjacent to alder stands in the winegrowing region of Rheinhessen in September 2020. Symptoms comprised leaf rolling and discoloration, incomplete lignification, black pustules on shoots, dried inflorescences and shriveled berries. Diseased shoots were black and necrotic in December. Leaf midribs were sampled for total DNA extraction. The phytoplasma 16S rRNA gene was amplified by generic primers R16F2/R2-mod followed by a nested PCR using 16Sr(V) group-specific primers R16(V)F1/R1, and primers R16(I)F1/R1 (Lee et al. 1995) to detect 'Candidatus Phytoplasma solani', associated with BN. While BN was detected in seven vines, one sample tested positive for 16SrV phytoplasma. This result was confirmed by triplex real-time Taq-Man assay based on rpl14 gene sequences (IPADLAB), by multiplex real-time PCR of map locus as well as by Loop-mediated isothermal amplification (LAMP) according to the EPPO diagnostic standard PM 7/079(2) (EPPO 2016). PCR-products of the map- and the vmpA-Gene (Malembic-Maher et al., 2020) were sequenced and compared to reference sequences to distinguish between FD- and non-FD genotypes. The isolate from the diseased vine exhibited 100% identity with map-M38 (Accession No. LT221933), a genotype of the map-FD2 cluster. The same genotype was detected in A. glutinosa and Allygus spp. sampled at the infested site. A 234 bp sequence of the first repeat of the vmpA-gene showed 100% identity with the S. titanus transmitted isolate FD-92 (Accession No. LN680870) of the vmpA-II cluster. It can be concluded, that the 16SrV-isolate detected in a symptomatic grapevine is infected by FD and not PGY. This is the first report of FD in a vineyard in Germany. The infected vine of cv. Silvaner was 25 years old. While infected planting material is an unlikely source of the infection, a transmission of FDp from alder is highly probable. Finding a single FD-infection after several years of testing implies a low risk originating from the wild compartment, but the approach of the vector S. titanus justifies further monitoring activities. The infected vine was eradicated.

As the period for field trials on grapevine is limited, we designed a laboratory test system to evaluate the effectiveness of selected insecticides against spotted-wing Drosophila (SWD), Drosophila suzukii, on different types of grape berries all year round. Tests were undertaken during winter and early spring with table grapes of different purchased varieties according to their seasonal availability and with wine grapes from experimental field plots in autumn. In preliminary experiments, we defined parameters for a standard laboratory test system for screening the effectiveness of several formulated insecticides in two different experimental set-ups: i) application before confining adults with berries and ii) application after confining adult D. suzukii with berries. These approaches allowed us to determine the contact activity of the products on adult D. suzukii or the impact on the larval development until the emergence of adult flies. The developed test system is suitable for screening substances with diverse types of activity on different grape types. In a second step, we combined laboratory bioassays with field applications in a semi-field persistence study and lastly we installed a randomized field plot in order to compare the effectiveness of selected insecticides in the laboratory and under field conditions. In all cases, the products Karate Zeon and SpinTor proved most efficacious in their contact mortality or as oviposition deterrents, while Mospilan SG and Coragen exhibited a good larvicidal activity. However, important disagreements occurred for the efficacy of currently authorized insecticides among laboratory, semi-field and practical field applications. The transferability of laboratory results into the field is discussed.

The phytopathogenic, cell-wall-less phytoplasmas exhibit a dual life cycle: they multiply in the phloem of their host plant and in the body of their insect vector. Their membrane proteins are in direct contact with both hosts and are supposed to play a crucial role in the phytoplasma spread within the plant as well as by the insect vector. Three types of nonhomologous but highly abundant and immunodominant membrane proteins (IDP) have been identified within the phytoplasmas: Amp, IdpA, and Imp. Although recent results indicate that Amp is involved in vector specificity interacting with insect proteins such as actin, little is known about the interaction of IDP with the plant. We could demonstrate that transiently expressed Imp of 'Candidatus Phytoplasma mali' as well as the Imp without transmembrane domain (Imp▴Tm) bind with plant actins in vivo. Moreover, in vitro co-sediment and binding assays showed that Escherichia coli-expressed recombinant Imp▴Tm-His binds to both G- and F-actins isolated from rabbit muscle. Transgenic plants expressing Imp- or Imp▴Tm-green fluorescent protein did not exhibit any remarkable change of phenotype compared with the wild-type plant. These results indicate that Imp specifically binds to plant actin and a role of Imp-actin binding in phytoplasma motility is hypothesized.

European stone fruit yellows (ESFY) is the common name of several economically important decline diseases of stone fruits (Prunus spp.) in Europe. A decline by apoplexy was first reported for apricot in France and for Japanese plum in Italy. Since then, several diseases have been described as chlorotic leaf roll of apricot, leptonecrosis and decline of Japanese plum, peach yellows and peach decline of peach, Molières disease of sweet cherry and European plum, and other diseases that include those affecting almond and flowering cherry. Molecular studies revealed that all these diseases are caused by a relatively homogeneous organism, the ESFY phytoplasma.

Pear decline (PD) is one of the most important diseases of pear. It is induced by a phytoplasma and was first described in some detail in British Columbia. Only a few years later, PD was observed in central Washington and subsequently spread further south to Oregon and was found in California in 1957. There is a clear link between the occurrence of the disease and the spread of pear psylla (Cacopsylla pyricola) along the Pacific coast. Analysis has revealed that PD is induced by a distinct phytoplasma that is closely related to the apple proliferation (AP) and to the European stone fruit yellows (ESFY) agents.

'Candidatus Phytoplasma prunorum' is an important prokaryotic pathogen that infects stone fruits in Europe. It is known to cause several economically relevant disorders of Prunus spp. which are collectively referred to as European stone fruit yellows (ESFY). This organism is phylogenetically closely related to other important fruit trees pathogens such as apple proliferation (AP), pear decline (PD) and peach yellow leaf roll (PYLR) agents. Together they form a distinct phylogenetic cluster, the AP or 16SrX group. Like the other fruit tree phytoplasmas of the AP group, 'Ca. P. prunorum' exhibits a high host specificity. In nature, this pathogen has been reported to infect only plants in the genus Prunus and to be transmitted by one insect vector species, the psyllid (Psyllidae) Cacopsylla pruni. Also, 'Ca. P. prunorum' includes strains which greatly differ in virulence. This review summarizes the current knowledge of 'Ca. P. prunorum' with emphasis on advances that have been made during the last two decades in understanding molecular and epidemiological aspects. Prospects for disease management and future research which could provide insights into the largely unknown mechanisms involved in pathogenicity of 'Ca. P. prunorum', are also critically discussed.

A real-time PCR assay for the quantification of Ca. Phytoplasma prunorum has been established which combines the specificity of detection with a low cost method of quantitative PCR. The assay uses the specific primers ECA1/ECA2 with a SYBR Green I protocol. A gene fragment of Ca. P. prunorum with the target of the primers has been cloned and is used as standard for quantification by the standard curve method. The assay has been successfully applied to measure the concentration of Ca. P. prunorum in insects as well as in different kinds of plant samples. Keywords : European stone fruit yellows, Cacopsylla pruni , resistance screening

Since 2000, surveys have been conducted in different stone fruit growing regions in South-western Germany to detect European stone fruit yellows (ESFY) disease in Germany. Each year visual inspections for typical symptoms of ESFY such as early budbreak in late winter and chlorotic leafroll in summer have been done on different Prunus species. Branch samples of all trees with typical symptoms as well as randomized samples from trees with doubtful symptoms have been taken in summer and analysed for infection with Candidatus Phytoplasma prunorum via PCR using specific primers ECA1/ECA2. The pathogen could be detected in the regions Rheinland, Rheinhessen, Vorder- and Sudpfalz and Baden and was present in all cultivated Prunus species: P. armeniaca, P. persica, A domestica and P. amygdalus. For apricots, more than 80% of the samples were infected while peach and European plum were less affected. Trees of P. armeniaca with typical symptoms showed up to 90% correlation with the presence of the phytoplasma but also a high percentage of trees with doubtful symptoms were highly infected. For P. persica, symptom specification was less pronounced. Almost no infection was found in the wild Prunus species P. spinosa and P. cerasifera. In contrast, regular psyllid captures on all different Prunus species gave high populations of Cacopsylla pruni on P. spinosa and P. cerasifera while only few individuals were collected from cultivated orchards. The natural infection rate of field collected C. pruni was between 2 and 3%. Transmission trials under controlled conditions showed the capability of C. pruni to transmit the phytoplasma to healthy test plants and proved that C. pruni is also a vector of Candidatus Phytoplasma prunorum in Germany.

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(TM) 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.

The transmission of apple proliferation (AP) phytoplasmas by psyllid vectors was investigated in greenhouse trials. Overwintering adults of Cacopsylla picta were captured from March to May in different orchards in Southwestern Germany. Groups of 5 to 30 individuals were caged for 2 to 4 weeks on apple seedlings or healthy micropropagated apple plants. Dead psyllids were collected and tested individually by PCR for AP phytoplasma infection. Leaf midribs of the test plants were sampled 2 to 3 months after inoculation feeding and were tested by PCR for AP phytoplasma infection. In 2002, 5 out of 11 test plants inoculated with overwintering adults of C. picta became AP phytoplasma-infected. Eleven individuals of C. picta collected from infected test plants were PCR-positive whereas all individuals collected from non-infected test plants were AP phytoplasma-negative. The transmission capability of the springtime generation of C picta was tested in an other experimental layout: young individuals of C. picta from breedings on healthy apple plants were first fed on AP-infected micropropagated test plants and then transferred to healthy test plants. In 2002, one out of 5 test plants were tested positive by PCR and showed AP-symptoms 6 months after inoculation. Fourteen individuals of the springtime generation of C. picta collected on this positive test plant were AP phytoplasma-positive. In 2003 we found 26 young C. picta positive by PCR and 3 PCR-positive test plants with AP-typical symptoms. These results confirm that C. picta is an important vector of AP phytoplasmas in Italy and Germany and give evidence that transmission occurs as well with overwintering adults as with individuals of the springtime generation.

PCR-RFLP using apple proliferation (AP) phytoplasma-specific primers has been employed to study the distribution of AP phytoplasma subtypes in commercial apple orchards as well as in abandoned or scattered orchards in a local fruit growing region of Palatinate in Southwest Germany. More than 50 samples from symptomatic trees were analysed. The AP phytoplasma subtype AP was found in 72% of the samples whereas the subtypes AT-2 and AT-1 were detected only in 16% and 12% of the samples, respectively. Important differences became evident regarding the distribution of the subtypes in different kinds of orchards. The AP subtype was exclusively found in commercial orchards. In abandoned or scattered orchards the AT-1 subtype was predominant. The latter subtype was never detected in commercial orchards. The subtype AT-2 showed an intermediate distribution and was found in both kinds of orchards. Eleven per cent of the samples from commercial orchards were of AT-2 subtype and 36% of the samples from abandoned or scattered orchards. The predominance of the AP subtype is in accordance with the previously reported European distribution of the subtypes. Our data indicate that the AP subtype either spreads more efficiently by the insect vector or multiplies more efficiently in the plant.

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.