The Effect of the Anticipated Nuclear Localization Sequence of ‘Candidatus Phytoplasma mali’ SAP11-like Protein on Localization of the Protein and Destabilization of TCP Transcription Factor

SAP11 is an effector protein that has been identified in various phytoplasma species. It localizes in the plant nucleus and can bind and destabilize TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors. Although SAP11 of different phytoplasma species share similar activities, their protein sequences differ greatly. Here, we demonstrate that the SAP11-like protein of ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) strain PM19 localizes into the plant nucleus without requiring the anticipated nuclear localization sequence (NLS). We show that the protein induces crinkled leaves and siliques, and witches’ broom symptoms, in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to six members of class I and all members of class II TCP transcription factors of A. thaliana in yeast two-hybrid assays. We also identified a 17 amino acid stretch previously predicted to be a nuclear localization sequence that is important for the binding of some of the TCPs, which results in a crinkled leaf and silique phenotype in transgenic A. thaliana. Moreover, we provide evidence that the SAP11-like protein has a destabilizing effect on some TCPs in vivo.

Study of the distribution of phytoplasmosis and fusariosis in certain areas of the Moscow region

In this work, we studied the distribution of especially dangerous phytopathogens - the causative agent of Apple proliferation (Candidatus Phytoplasma mali) and causative agents of root rot (Fusarium oxysporum, F. sambucinum, F. culmorum) in three districts of the Moscow region.

Surveying Potential Vectors of Apple Proliferation Phytoplasma: Faunistic Analysis and Infection Status of Selected Auchenorrhyncha Species

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.

Identification of Plant DNA in Adults of the Phytoplasma Vector Cacopsylla picta Helps Understanding Its Feeding Behavior

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 phytopathogen ‘Candidatus Phytoplasma mali’ alters apple tree phloem composition and affects oviposition behavior of its vector Cacopsylla picta

Apple proliferation disease is caused by the phloem-dwelling bacterium ‘Candidatus Phytoplasma mali’, inducing morphological changes in its host plant apple, such as witches’ broom formation. Furthermore, it triggers physiological alterations like emission of volatile organic compounds or phytohormone levels in the plant. In our study, we assessed phytoplasma-induced changes in the phloem by sampling phloem sap from infected and non-infected apple plants. In infected plants, the soluble sugar content increased and the composition of phloem metabolites differed significantly between non-infected and infected plants. Sugar and sugar alcohol levels increased in diseased plants, while organic and amino acid content remained constant. As ‘Ca. P. mali’ is vectored by the phloem-feeding insect Cacopsylla picta (Foerster, 1848), we assessed whether the insect–plant interaction was affected by ‘Ca. P. mali’ infection of the common host plant Malus domestica Borkh. Binary-choice oviposition bioassays between infected and non-infected apple leaves revealed C. picta’s preference for non-infected leaves. It is assumed and discussed that the changes in vector behavior are attributable to plant-mediated effects of the phytoplasma infection.

Effect of Daytime and Tree Canopy Height on Sampling of Cacopsylla melanoneura, a ‘Candidatus Phytoplasma mali’ Vector

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.

Temporal Dynamics of ‘Ca. Phytoplasma mali’ Load in the Insect Vector Cacopsylla melanoneura

The transmission of phytoplasmas is the result of an intricate interplay involving pathogens, insect vectors and host plants. Knowledge of the vector’s competence during its lifespan allows us to define more sustainable well-timed control strategies targeted towards the most worrisome life stages. We investigated the temporal dynamics of ‘Candidatus Phytoplasma mali’ load in Cacopsylla melanoneura in the different developmental stages in Northwest Italy. The phytoplasma load in the vector was evaluated in overwintering adults, nymphs and newly emerged adults after different acquisition access periods. Moreover, we followed the multiplication of the phytoplasma during the aestivation and the overwintering period on conifers. Our results confirmed the ability of remigrants to retain the phytoplasma until the end of winter. We also highlighted the high acquisition efficiency and vector competence, based on phytoplasma load, of nymphs and newly emerged adults. Therefore, particular attention should be paid to the management of overwintered C. melanoneura as soon as they return to the orchards, but also to newly emerged adults, particularly in orchards with a high infection rate and when the migration to conifers is delayed.

The anticipated potential nuclear localization sequence of ‘Candidatus Phytoplasma mali’ SAP11-like protein is required for TCP binding but not for transport into the nucleus

The plant pathogen ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) is the causing agent of apple proliferation that leads to heavy damage in apple production all over Europe. To identify and analyze effector proteins of plant pathogens is an important strategy in plant disease research. Here, we report that the SAP11-like protein of ‘Ca. P. mali’ induces crinkled leaves and siliques and witches’ broom symptoms in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to 6 members of class I and all members of class II TCP (TEOSINE BRANCHES/ CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of A. thaliana in yeast two-hybrid assays. Moreover, we demonstrate that the protein localizes actively into the plant nucleus without requiring the nuclear leader sequence (NLS). We also identified a 17 amino acid stretch previously predicted to be a nuclear leader sequence that is important for the binding of some of the TCPs and also responsible for the crinkled leaf and silique phenotype in transgenic A. thaliana.