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Phytoplasma diseases pose a substantial threat to diverse crops of agricultural importance. Management measures are usually implemented only after the disease has already occurred. Early detection of such phytopathogens, prior to disease outbreak, has rarely been attempted, but would be highly beneficial for phytosanitary risk assessment, disease prevention and mitigation. In this study, we present the implementation of a recently proposed proactive disease management protocol (DAMA: Document, Assess, Monitor, Act) for a group of vector-borne phytopathogens. We used insect samples collected during a recent biomonitoring program in southern Germany to screen for the presence of phytoplasmas. Insects were collected with malaise traps in different agricultural settings. DNA was extracted from these mass trap samples and subjected to PCR-based phytoplasma detection and mitochondrial cytochrome c oxidase subunit I (COI) metabarcoding. Phytoplasma DNA was detected in two out of the 152 insect samples analyzed. Phytoplasma identification was performed using iPhyClassifier based on 16S rRNA gene sequence and the detected phytoplasmas were assigned to ‘Candidatus Phytoplasma asteris’-related strains. Insect species in the sample were identified by DNA metabarcoding. By using established databases, checklists, and archives, we documented historical associations and records of phytoplasmas and its hosts in the study region. For the assessment in the DAMA protocol, phylogenetic triage was performed in order to determine the risk for tri-trophic interactions (plant–insect–phytoplasma) and associated disease outbreaks in the study region. A phylogenetic heat map constitutes the basis for risk assessment and was used here to identify a minimum number of seven leafhopper species suggested to be monitored by stakeholders in this region. A proactive stance in monitoring changing patterns of association between hosts and pathogens can be a cornerstone in capabilities to prevent future phytoplasma disease outbreaks. To the best of our knowledge, this is the first time that the DAMA protocol has been applied in the field of phytopathology and vector-borne plant diseases.

Apple proliferation (AP) is one of the economically most important diseases in European apple cultivation. The disease is caused by the cell-wall-less bacterium ’ Candidatus Phytoplasma mali’, which is transmitted by Cacopsylla picta (Foerster) and Cacopsylla melanoneura (Foerster) (Hemiptera: Psylloidea). In South Tyrol (Italy), severe outbreaks were documented since the 1990s. Infestation rates of AP do not always correlate with the population densities of the confirmed vectors, implying the presence of other, so far unknown, hemipterian vectors. By elucidating the species community of Auchenorrhyncha (Insecta: Hemiptera) at a regional scale, more than 31,000 specimens were captured in South Tyrolean apple orchards. The occurrence of 95 species was confirmed, whereas fourteen species are new records for this territory. Based on the faunistical data, more than 3600 individuals out of 25 species were analyzed using quantitative PCR to assess the presence of AP phytoplasma. The pathogen was sporadically detected in some individuals of different species, for example in Stictocephala bisonia Kopp and Yonk (Hemiptera: Membracidae). However, the concentration of phytoplasma was much lower than in infected C. picta and C. melanoneura captured in the same region, confirming the role of the latter mentioned psyllids as the main insect vectors of AP- phytoplasma in South Tyrol.

Apple proliferation is an economically important disease and a threat for commercial apple cultivation. The causative pathogen, the bacterium ‘Candidatus Phytoplasma mali’, is mainly transmitted by Cacopsylla picta, a phloem-feeding insect that develops on the apple tree (Malus spp.). To investigate the feeding behavior of adults of the phytoplasma vector Cacopsylla picta in more detail, we used deep sequencing technology to identify plant-specific DNA ingested by the insect. Adult psyllids were collected in different apple orchards in the Trentino-South Tyrol region of northern Italy. DNA from the whole body of the insect was extracted and analyzed for the presence of plant DNA by performing PCR with two plant-specific primers that target the chloroplast regions trnH-psbA and rbcLa. DNA from 23 plant genera (trnH) and four plant families (rbcLa) of woody and herbaceous plant taxa was detected. Up to six and three plant genera and families, respectively, could be determined in single specimens. The results of this study contribute to a better understanding of the feeding behavior of adult Cacopsylla picta.

The psyllids Cacopsylla melanoneura and Cacopsylla picta reproduce on apple (Malus × domestica) and transmit the bacterium ‘Candidatus Phytoplasma mali’, the causative agent of apple proliferation. Adult psyllids were collected by the beating-tray method from lower and upper parts of the apple tree canopy in the morning and in the afternoon. There was a trend of catching more emigrant adults of C.melanoneura in the morning and in the lower part of the canopy. For C.melanoneura remigrants, no differences were observed. The findings regarding the distribution of adults were reflected by the number of nymphs collected by wash-down sampling. The density of C.picta was too low for a statistical analysis. The vector monitoring and how it is commonly performed, is suitable for estimating densities of C.melanoneura. Nevertheless, above a certain temperature threshold, prediction of C.melanoneura density might be skewed. No evidence was found that other relatively abundant psyllid species in the orchard, viz. Baeopelma colorata, Cacopsylla breviantennata, Cacopsylla brunneipennis, Cacopsylla pruni and Trioza urticae, were involved in ‘Candidatus Phytoplasma mali’ transmission. The results of our study contribute to an advanced understanding of insect vector behavior and thus have a practical impact for an improved field monitoring.

Effector proteins play an important role in the virulence of plant pathogens such as phytoplasma, which are the causative agents of hundreds of different plant diseases. The plant hosts comprise economically relevant crops such as apples (Malus × domestica), which can be infected by ‘Candidatus Phytoplasma mali’ (P. mali), a highly genetically dynamic plant pathogen. As the result of the genetic and functional analyses in this study, a new putative P. mali effector protein was revealed. The so-called “Protein in Malus Expressed 2” (PME2), which is expressed in apples during P. mali infection but not in the insect vector, shows regional genetic differences. In a heterologous expression assay using Nicotiana benthamiana and Nicotiana occidentalis mesophyll protoplasts, translocation of both PME2 variants in the cell nucleus was observed. Overexpression of the effector protein affected cell integrity in Nicotiana spp. protoplasts, indicating a potential role of this protein in pathogenic virulence. Interestingly, the two genetic variants of PME2 differ regarding their potential to manipulate cell integrity. However, the exact function of PME2 during disease manifestation and symptom development remains to be further elucidated. Aside from the first description of the function of a novel effector of P. mali, the results of this study underline the necessity for a more comprehensive description and understanding of the genetic diversity of P. mali as an indispensable basis for a functional understanding of apple proliferation disease.