Apple Proliferation Research Articles

‘Candidatus Phytoplasma mali’ SAP11-Like protein modulates expression of genes involved in energy production, photosynthesis, and defense in Nicotiana occidentalis leaves

Background‘Candidatus Phytoplasma mali’, the causal agent of apple proliferation disease, exerts influence on its host plant through various effector proteins, including SAP11CaPm which interacts with different TEOSINTE BRANCHED1/ CYCLOIDEA/ PROLIFERATING CELL FACTOR 1 and 2 (TCP) transcription factors. This study examines the transcriptional response of the plant upon early expression of SAP11CaPm. For that purpose, leaves of Nicotiana occidentalis H.-M. Wheeler were Agrobacterium-infiltrated to induce transient expression of SAP11CaPm and changes in the transcriptome were recorded until 5 days post infiltration.ResultsThe RNA-seq analysis revealed that presence of SAP11CaPm in leaves leads to downregulation of genes involved in defense response and related to photosynthetic processes, while expression of genes involved in energy production was enhanced.ConclusionsThe results indicate that early SAP11CaPm expression might be important for the colonization of the host plant since phytoplasmas lack many metabolic genes and are thus dependent on metabolites from their host plant.

Vom Chaos zur Klarheit. Die Neuordnung der Apfel TCP Gen-Benennung

Apple Proliferation (AP) is a widespread disease affecting apple orchards throughout Europe, including South Tyrol.The disease is associated with a phytoplasma, 'Candidatus Phytoplasma mali' ('Ca. P. mali'), which manipulates the plant's physiological processes through the secretion of small peptides called effectors. One well-studied phytoplasma effector is SAP11, which targets Teosinte Branched 1/Cycloidea/Proliferating Cell Factor 1 (TCP) genes in multiple plant species, including apple. TCP genes encode plant-specific transcription factors involved in various biological processes, such as growth, development, and responses to stimuli. The identification and naming of TCP genes have been inconsistent, leading to confusion and redundancy in sequence databases. In apple, 52 TCP genes (MdTCP) were identified in 2014 on the 2010 assembly of the apple genome, which is now considered outdated. To address this issue, a comprehensive investigation was conducted to identify and name TCP genes in apple using the high-quality genome assembly, GDDH13v1.1. Existing TCP sequences were aligned with the genome excluding redundant, fragmented, and non-TCP sequences. The revised set comprised 40 unique MdTCPs, including three novel genes. To establish a standardised nomenclature, each MdTCP was BLASTedagainst the Arabidopsis thaliana gene database, in order to identify the best hits for each MdTCP. The MdTCP genes were then renamed, incorporating the letter "a" or "b" to differentiate between MdTCPs showing homology to the same AtTCP, and also between the existing and proposed nomenclatures. The present study highlights the need for clarity and organisation in sequence databases, especially in respect of TCP genes. The presence of redundant and fragmented sequences in databases complicates accurate identification and annotation of genes. By providing a comprehensive and standardised nomenclature system, we aim to enhance the coherence and interoperability of future TCP gene research in plant and crop science. In our view, this work will serve as a valuable resource for researchers studying TCP genes in apple and will provide insights into the evolutionary dynamics and functional roles of TCP genes in plants.

Phytoplasma-Induced Leaf Reddening as a Monitoring Symptom of Apple Proliferation Disease with Regard to the Development of Remote Sensing Strategies

Apple proliferation (AP) is an economically important disease in many apple-growing regions caused by ‘Candidatus Phytoplasma mali’ which is spread by migrating psyllid vectors on a regional scale. As infected trees in orchards are the only inoculum source, the early eradication of those trees is one of the most efficient strategies to prevent further spread of AP. Remote sensing is a promising rapid and cost-effective tool to identify infected trees on a regional scale. AP-induced premature leaf reddening was evaluated as a reliable symptom for remote sensing by monitoring more than 20,000 trees in 68 different orchards with 20 representative cultivars from 2019 to 2022 in a highly AP-affected region in Southwest Germany. Specific AP symptoms were almost 100% correlated with molecular detection of ‘Ca. P. mali’ and these specific symptoms were almost 100% correlated with leaf reddening. ‘Ca. P. mali’ was detected in 71–97% of trees which showed partial or entire reddening without any other AP symptom. Experimental and field data showed that reddening was induced by cold night and warm day temperatures (about 5 °C vs. 20 °C) in September. Quantification of the phytoplasma by real-time PCR showed no correlation with the intensity of reddening in the leaf. PCR-RFLP subtyping revealed no influence of different ‘Ca. P. mali’ strains on the symptom expression. In conclusion, leaf reddening in late September/early October was a reliable symptom useful for remote sensing of AP.

Measuring 3D tree imbalance of plant models using graph-theoretical approaches

Imbalance in the 3D structure of plants can be an important indicator of insufficient light or nutrient supply, as well as excessive wind, (formerly present) physical barriers, neighbor or storm damage. It can also be a simple means to detect certain illnesses, since some diseases like the apple proliferation disease, an infection with the barley yellow dwarf virus or plant canker can cause abnormal growth, like “witches' brooms” or burls, resulting in a deviating 3D plant architecture. However, quantifying imbalance of plant growth is not an easy task, and it requires a mathematically sound 3D model of plants to which imbalance indices can be applied. Current models of plants are often based on stacked cylinders or voxel matrices and do not allow for measuring the degree of 3D imbalance in the branching structure of the whole plant.On the other hand, various imbalance indices are readily available for so-called graph-theoretical trees and are frequently used in areas like phylogenetics and computer science. While only some basic ideas of these indices can be transferred to the 3D setting, graph-theoretical trees are a logical foundation for 3D plant models that allow for elegant and natural imbalance measures.In this manuscript, our aim is thus threefold: We first present a new graph-theoretical 3D model of plants and discuss desirable properties of imbalance measures in the 3D setting. We then introduce and analyze eight different 3D imbalance indices and their properties. Thirdly, we illustrate all our findings using a data set of 63 bush beans. Moreover, we implemented all our indices in the publicly available R-software package treeDbalance accompanying this manuscript. Using this software package, all presented 3D imbalance indices can be computed in linear time (depending on the size of the 3D plant model), and the package also provides an implementation of the algorithm to obtain a perfectly balanced version of a given 3D plant model (also in linear time).

Clarithromycin Suppresses Apple Proliferation Phytoplasma in Explant Cultures.

Apple proliferation, caused by 'Candidatus Phytoplasma mali', is one of the most important economic threats in the field of apple production. Especially at a young age, infected trees can be affected by excessive bud proliferation and general decline. The fruit quality is also significantly reduced by this disease. To investigate treatment options, we applied a clarithromycin chemotherapy to infected in vitro cultures of 'Golden Delicious'. With increasing concentrations of clarithromycin in the media, the phytoplasma load decreased rapidly after one month of treatment, but phytotoxicity led to a pronounced mortality at 40 mg/L, which was the highest dose used in our experiment. Out of 45 initial explants, we obtained one negative mericlone and two mericlones with a concentration of phytoplasma DNA at the detection limit of PCR. The culture propagated from the mericlone that tested negative remained phytoplasma-free after 18 months of subculturing. Our results suggest the applicability of macrolide antibiotics against phytoplasma infections in vitro; however, it might be challenging to find the threshold zone where the concentration is sufficient for pathogen elimination, but not lethal for the plant material of different cultivars.

Multiple factors driving the acquisition efficiency of apple proliferation phytoplasma in Cacopsylla melanoneura

Phytoplasmas are bacterial pathogens located in the plant’s phloem that are responsible for several plant diseases and are mainly transmitted by phloem-sucking insects. Apple proliferation (AP) is an economically important disease associated with the presence of ‘Candidatus Phytoplasma mali’ which is transmitted by two psyllid species. While Cacopsylla picta is a vector in different regions, the vector efficiency of C. melanoneura varies between different populations. This species is considered the main AP vector in Northwestern Italy but plays a minor role in Northeastern Italy and other European regions. To investigate whether the psyllid and/or the phytoplasma subtype drive the phytoplasma acquisition in C. melanoneura, a phytoplasma acquisition experiment was set up using single mating couples of overwintered individuals from different psyllid populations and phytoplasma subtypes. All analyzed insect populations acquired phytoplasma, but with different efficiencies and concentrations. The main factors driving the acquisition were the phytoplasma subtype and its concentration in the leaves of the infected trees together with the psyllid lineage. The phytoplasma concentration in the psyllids was again influenced by the phytoplasma subtype, the psyllid lineage and the region of origin, whereas the phytoplasma concentration in the leaves and the psyllid haplotype defined with the cytochrome oxidase I gene had only a minor impact on the phytoplasma concentration. This is the first study evaluating the roles of both the psyllid haplotype and the phytoplasma subtype on the acquisition process and highlights the importance of C. melanoneura as an additional AP vector.

Identification of spectral ranges that contribute to phytoplasma detection in apple trees – A step towards an on-site method

'Candidatus Phytoplasma mali' is the bacterial agent associated with Apple Proliferation, a disease that causes high economic losses in affected commercial apple growing regions. The identification of the disease is carried out by visual inspection performed by skilled professionals in the orchards. To confirm an infection, costly molecular laboratory methods must be applied. Furthermore, both methods are very time-consuming. Here, we analysed the potential of a non-destructive method using in-field measurements to differentiate infected from non-infected apple trees (Malus domestica) based on spectral signatures of fresh leaves. By using multivariate statistics, we were able to distinguish infected from non-infected trees and identified the wavelengths relevant for the differentiation. Factors affecting the differentiation performance were the sampling date and bacterial colonization behaviour.

Sanitation of Apple Cultivars from AP Phytoplasma and ApMV and ACLSV Viruses Using In Vitro Culture and Cryo-Knife Therapy in Liquid Nitrogen

Systemic infections with phytoplasmas and viruses threaten the production of healthy plant material under the fruit species certification system. We tested the possibility of sanitation using in vitro culture and cryotherapy. The starting material of the cultivars Golden Delicious (clones A and B), Virginia Crab, and Panenské zlepšené was taken from in vivo plants that tested positive for apple proliferation phytoplasma. The Táborita cultivar was obtained from already established in vitro cultures that had tested positive for apple proliferation phytoplasma, apple mosaic virus, and apple chlorotic leaf spot virus. Cultivars Golden Delicious A, Virginia Crab, and Panenské zlepšené were sanitated from the phytoplasma in the first step, i.e., by sterilization and a subsequent transfer to in vitro conditions. Golden Delicious B remained infected with the phytoplasma, and both viruses, after the in vitro culture phase and together with Táborita, were subjected to cryotherapy by vitrification. In Golden Delicious B, three out of thirteen initial shoot tips regenerated after a liquid nitrogen treatment. Four mericlones were regenerated from 10 initial cryopreserved shoot tips of Táborita. None of the three pathogens were detected by PCR in the regenerated Golden Delicious B mericlones. On the contrary, in the case of Táborita, infection with all the pathogens was detected after regeneration. The results obtained indicate the potential applicability of in vitro cultivation techniques or, if necessary, subsequent cryopreservation as a method for sanitizing against systemic microbial contamination. However, further research on the relationship between pathogens and specific genotypes is needed.

Orientus ishidae (Hemiptera: Cicadellidae): Biology, Direct Damage and Preliminary Studies on Apple Proliferation Infection in Apple Orchard.

The mosaic leafhopper, Orientus ishidae (Matsumura), is an Asian species widespread in Europe that can cause leaf damage in wild trees and transmit disease phytoplasmas to grapevines. Following an O. ishidae outbreak reported in 2019 in an apple orchard in northern Italy, the biology and damage caused by this species to apples were investigated during 2020 and 2021. Our studies included observations on the O. ishidae life cycle, leaf symptoms associated to its trophic activity, and its capability to acquire "Candidatus Phytoplasma mali," a causal agent of Apple Proliferation (AP). The results indicate that O. ishidae can complete the life cycle on apple trees. Nymphs emerged between May and June, and adults were present from early July to late October, with the peak of flight between July and early August. Semi-field observations allowed for an accurate description of leaf symptoms that appeared as a distinct yellowing after a one-day exposure. In field experiments, 23% of the leaves were found damaged. In addition, 16-18% of the collected leafhoppers were found carrying AP phytoplasma. We conclude that O. ishidae has the potential to be a new apple tree pest. However, further studies are required to better understand the economic impact of the infestations.

Screening of European stone fruit yellows, apple proliferation and pear decline diseases in fruit nurseries with focus on latent infection

Screening of European stone fruit yellows, apple proliferation and pear decline diseases in fruit nurseries with focus on latent infection

The 'Candidatus Phytoplasma mali' effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection.

'Candidatus Phytoplasma mali', is a bacterial pathogen associated with the so-called apple proliferation disease in Malus × domestica. The pathogen manipulates its host with a set of effector proteins, among them SAP11CaPm, which shares similarity to SAP11AYWB from 'Candidatus Phytoplasma asteris'. SAP11AYWB interacts and destabilizes the class II CIN transcription factors of Arabidopsis thaliana, namely AtTCP4 and AtTCP13 as well as the class II CYC/TB1 transcription factor AtTCP18, also known as BRANCHED1 being an important factor for shoot branching. It has been shown that SAP11CaPm interacts with the Malus × domestica orthologues of AtTCP4 (MdTCP25) and AtTCP13 (MdTCP24), but an interaction with MdTCP16, the orthologue of AtTCP18, has never been proven. The aim of this study was to investigate this potential interaction and close a knowledge gap regarding the function of SAP11CaPm. A Yeast two-hybrid test and Bimolecular Fluorescence Complementation in planta revealed that SAP11CaPm interacts with MdTCP16. MdTCP16 is known to play a role in the control of the seasonal growth of perennial plants and an increase of MdTCP16 gene expression has been detected in apple leaves in autumn. In addition to this, MdTCP16 is highly expressed during phytoplasma infection. Binding of MdTCP16 by SAP11CaPm might lead to the induction of shoot proliferation and early bud break, both of which are characteristic symptoms of apple proliferation disease.

Investigating the microbial community of Cacopsylla spp. as potential factor in vector competence of phytoplasma.

Phytoplasmas are obligatory intracellular bacteria that colonize the phloem of many plant species and cause hundreds of plant diseases worldwide. In nature, phytoplasmas are primarily transmitted by hemipteran vectors. While all phloem-feeding insects could in principle transmit phytoplasmas, only a limited number of species have been confirmed as vectors. Knowledge about factors that might determine the vector capacity is currently scarce. Here, we characterized the microbiomes of vector and non-vector species of apple proliferation (AP) phytoplasma 'Candidatus Phytoplasma mali' to investigate their potential role in the vector capacity of the host. We performed high-throughput 16S rRNA metabarcoding of the two principal AP-vectors Cacopsylla picta and Cacopsylla melanoneura and eight Cacopsylla species, which are not AP-vectors but co-occur in apple orchards. The microbiomes of all species are dominated by Carsonella, the primary endosymbiont of psyllids and a second uncharacterized Enterobacteriaceae endosymbiont. Each Cacopsylla species harboured a species-specific phylotype of both symbionts. Moreover, we investigated differences between the microbiomes of AP-vector versus non-vector species and identified the predominant endosymbionts but also Wolbachia and several minor taxa as potential indicator species. Our study highlights the importance of considering the microbiome in future investigations of potential factors influencing host vector competence. We investigated the potential role of symbiotic bacteria in the acquisition and transmission of phytoplasma. By comparing the two main psyillid vector species of Apple proliferation (AP) phytoplasma and eight co-occurring species, which are not able to vector AP-phytoplasma, we found differences in the microbial communities of AP-vector and non-vector species, which appear to be driven by the predominant symbionts in both vector species and Wolbachia and several minor taxa in the non-vector species. In contrast, infection with AP-phytoplasma did not affect microbiome composition in both vector species. Our study provides new insights into the endosymbiont diversity of Cacopsylla spp. and highlights the importance of considering the microbiome when investigating potential factors influencing host vector competence.

Determination of the efficacy of endophytic fungi obtained from apple (Malus domestica) trees on apple proliferation phytoplasma disease (Candidatus Pyhtoplasma mali)

Amaç: Bu çalışmada Elma Çoklu Sürgün Fitoplazma Hastalığı (EÇSF) (Candidatus Pyhtoplasma mali)’nın biyolojik mücadelesinde endofitik fungusların etkinliğinin belirlenmesi amaçlanmıştır. Böylece pratikte kimyasal mücadele olanağı olmayan hastalık etmenine karşı farklı kontrol stratejileri geliştirilebilme olanağının sağlanması hedeflenmiştir. Yöntem ve Bulgular: EÇSF ve endofit fungus izolasyonları için toplam 50 farklı bahçe gezilerek fitoplazma testleri için 360 ağaçtan, endofit funguslar için ise hem sağlıklı hem de fitoplazma ile enfekteli 198 ağaçtan bitki örneği toplanmıştır. Fitoplazma açısından testlenen 360 örnekten 29 tanesi ‘Ca. P. mali’ ile enfekteli bulunmuştur. ‘Ca. P. mali’ ile enfekteli ve sağlıklı elma ağaçlarından endofit fungus türleri izole edilmiş, bu fungusların morfolojik ve moleküler tanısı ile birlikte patojenisite çalışmaları yapılmıştır. Sağlıklı elma ağaçlarından en yaygın olarak izole edilen endofit funguslardan Epicoccum nigrum, Aureobasidium pullulans, Ulocladium consortiale’nin EÇSF hastalığına karşı etkinlikleri incelenmiştir. Her bir fungus izolatının EÇSF hastalık yoğunluğunu farklı düzeylerde etkilediği, en yüksek etkinin A. pullulans’a ait olduğu ve EÇSF hastalık yoğunluğunu uygulama sonrası %34,18 etki değeri ile azalttığı belirlenmiştir. Genel Yorum: EÇSF hastalığının etkili bir kimyasal mücadelesinin bulunmaması ve etmenin karantina listesinde yer alması nedeniyle olası biyolojik mücadele uygulamaları oldukça önemlidir. EÇSF ile enfekteli ağaçlara inokule edilen 3 farklı endofit fungus türünden Aureobasidium pullulans’ın fitoplazma yoğunluğunu diğer 2 etmene göre daha fazla azaltmasından dolayı A. pullulans hastalıkla mücadelede kullanılabilecek biyolojik mücadele ajanı olabilir. Çalışmanın Önemi ve Etkisi: Bu çalışma ile biyolojik mücadele ajanı olan endofit fungusların elmalarda EÇSF hastalığı üzerine etkileri Türkiye’de ilk kez araştırılmış ve hastalığın kontrol stratejilerinin oluşturulmasında önemli ön bulgular elde edilmiştir.

Phytoplasma mali (apple proliferation)

Phytoplasma mali (apple proliferation)

Identification of 16SrXXX-A Phytoplasma Associated with Salt cedar (Tamarix chinensis Lour.) witches'-broom (SCWB) in Southern Xinjiang of China.

Salt cedar is an ornamental shrub/moderate tree species native to Asia and East Europe, and grows in salt-alkali soil, desert and other dry areas, which plays an important role in wind prevention and sand fixation as well as maintaining ecological balance. Salt cedar witches'-broom (SCWB), which was extremely pernicious to Salt cedar. It was first observed and reported in Xi'an, China in 2005 (Zhao et al.2005). Witches' broom symptoms were observed on 20 out of 150 (13.3%) salt plants surveyed from the Alar region and 10 out of 86 (11.6%) plants from the Akesu region in southern of Xinjiang in May 2020. The damaged plants compared with asymptomatic plants (Fig.1A), the major symptoms included branches clustered, intersegment shorten and coarsen, giving rise to the formation of clusters (Fig.1B). Total plant DNA was extracted from phloem tissues with asymptomatic symptoms and phloem tissues with witches'-broom symptoms by a CTAB-based DNA extraction method (Green et al.1999). The 16S rRNA gene and the phytoplasma universal primers P1/P7 and rpF1/rpR1 of the rp (ribosomal protein) gene were used for Polymerase chain reaction (PCR) amplification by using the extracted plant total DNA as the template. The PCR product was used as the template and the R16F2n/R16R2 prmer was used for nested PCR amplification of the 16S rRNA gene after the amplification was completed. The results show that no product was obtained in asymptomatic plants. When DNA samples from witches'-broom symptomatic plants were used as templates, fragments with lengths 1219 bp and 1174 bp, corresponding to 16S rRNA gene and rp gene, were obtained. 16S rRNA gene was sequenced and deposited in GenBank under accession number MW447513. BLAST analysis revealed that the partial 16S rRNA sequence of the phytoplasma associated with P. aphylla witches' broom showed highest sequence identity (99.67%) to salt cedar witches' broom phytoplasma, 'Candidatus Phytoplasma tamaricis' (Accession Number: FJ432664). Phylogenetic and molecular evolutionary analyses were conducted using MEGA-X (Kumar et al., 2018). Results showed taht the SCWB and 16S rXXX group's'Candidatus Phytoplasma tamaricis', (GenBank accession: FJ432664) have the highest affinity (Fig.2A). A virtual restriction fragment length polymorphism(RFLP) was done to determinethe subgroup ( Zhao et al. 2009). The 16S rDNA sequence from the Tamarix chinensis plant showed 99.3% similarity with that of the "Candidatus Phytoplasma tamaricis" reference strain (GenBank accession: FJ432664), suggesting that the phytoplasma in this study belongs to "Candidatus Phytoplasma tamaricis"-related strain. Therefore, it can be stated that SCWB belongs to the 16S rXXX group. The partial rp sequences only shared 84.74% sequence similarity with that of 'Candidatus Phytoplasma prunorum' (MG383523) of Apple proliferation group, a known subgroup 16S rX. Blast analysis based on the partial rp sequences showed that it shares less than 90% similarity with that of any known phytoplasma (Fig 2B), we suspect that this is due to a lack of sequenced rp gene sequences for the 16S rXXX group. To our knowledge, this is the first report of Salt Cedar Witches' Broom phytoplasma in Xinjiang province, China. As a consequence, we guess the SCWB phytoplasma rp gene belongs to 16S rXXX-rp group, which is also the first report about the 16SrXXX-rp group. Because SCWB1 is the only strain in the 16S rXXX group, and it is the representative strain of the 16S rXXX-A subgroup (Zhao et al. 2009). So, the SCWB disease we found in southern Xinjiang belongs to the 16S rXXX-A subgroup.

Detection of apple proliferation disease in Malus × domestica by near infrared reflectance analysis of leaves

In this study near infrared spectroscopical analysis of dried and ground leaves was performed and combined with a multivariate data analysis to distinguish 'Candidatus Phytoplasma mali' infected from non-infected apple trees (Malus × domestica). The bacterium is the causative agent of Apple Proliferation, one of the most threatening diseases in commercial apple growing regions. In a two-year study, leaves were sampled from three apple orchards, at different sampling events throughout the vegetation period. The spectral data were analyzed with a principal component analysis and classification models were developed. The model performance for the differentiation of Apple Proliferation diseased from non-infected trees increased throughout the vegetation period and gained best results in autumn. Even with asymptomatic leaves from infected trees a correct classification was possible indicating that the spectral-based method provides reliable results even if samples without visible symptoms are analyzed. The wavelength regions that contributed to the differentiation of infected and non-infected trees could be mainly assigned to a reduction of carbohydrates and N-containing organic compounds. Wet chemical analyses confirmed that N-containing compounds are reduced in leaves from infected trees. The results of our study provide a valuable indication that spectral analysis is a promising technique for Apple Proliferation detection in future smart farming approaches.

Pathogenicity against hemipteran vector insects of a novel insect pathogenic fungus from Entomophthorales (Pandora sp. nov. inedit.) with potential for biological control

A new but still unpublished entomopathogenic fungus (ARSEF13372) in the genus Pandora (Entomophthorales: Entomophthoraceae) was originally isolated from Cacopsylla sp. (Hemiptera: Psyllidae). Several species of the genus Cacopsylla vector phloem-borne bacteria of the genus ‘Candidatus Phytoplasma‘, which cause diseases in fruit crops such as apple proliferation, pear decline and European stone fruit yellows. To determine Pandora’s host range and biocontrol potential we conducted laboratory infection bioassays; Hemipteran phloem-feeding insects were exposed to conidia actively discharged from in vitro produced mycelial mats of standardized area. We documented the pathogenicity of Pandora sp. nov. to species of the insect families Psyllidae and Triozidae, namely Cacopsyllapyri L., C.pyricola (Foerster), C.picta (Foerster, 1848), C.pruni (Scopoli), C.peregrina (Foerster), and Trioza apicalis Foerster. The occurrence of postmortem signs of infection on cadavers within 10 days post inoculation proved that Pandora sp. nov. was infective to the tested insect species under laboratory conditions and significantly reduced mean survival time for C.pyri (summer form and nymph), C.pyricola, C.picta, C.pruni, C.peregrina and T.apicalis. Assessing a potential interaction between phytoplasma, fungus and insect host revealed that phytoplasma infection (‘Candidatus Phytoplasma mali’) of the vector C.picta and/or its host plant apple Malus domestica Borkh. did not significantly impact the survival of C.picta after Pandora sp. nov. infection. The results from infection bioassays were discussed in relation to Pandora sp. nov. host range and its suitability as biocontrol agent in integrated pest management strategies of psyllid pests, including vector species, in orchards.

Influence of ontogenetic and migration stage on feeding behavior of Cacopsylla picta on ‘Candidatus Phytoplasma mali’ infected and non-infected apple plants

The summer apple psyllid Cacopsylla picta (Foerster) is the vector of ‘Candidatus Phytoplasma mali’, the causal agent of apple proliferation disease (AP). During its phloem-feeding activities it transmits this biotrophic bacterium from infected to healthy apple trees (Malus domestica Borkh.) causing high economic losses. During its life cycle, C.picta performs two host switches: In summer, the new adult generation (emigrants) hatch on apples before they emigrate to their overwintering host conifers. The following spring, the overwintered adult generation (remigrants) remigrate into apple orchards for mating and oviposition. The preimaginal stages (nymphs) develop on apple. It is known that phytopathogen-induced changes in plant physiology can affect insect-plant-interactions. In 12 h recordings of electrical penetration graphs (EPG) it was assessed whether ‘Ca. P. mali’ infection of the plant affected probing and feeding behavior of the vector C.picta. Its life stage and the infection status of the host plant (and the interaction between these factors) significantly affected the first occurrence, duration and frequency of probing and feeding phases. On ‘Ca. P. mali’ infected plants, the phloem salivation phase occurred later than on non-infected plants. Even though all life stages fed both on phloem and xylem, significant differences were found in the frequency and duration of phloem and xylem ingestion phases. Nymphs spent the shortest time non-probing, earlier started the first leaf penetration and longer ingested xylem compared with adults. Further, phloem phases differed between migratory stages; remigrants had higher numbers of phloem ingestion events and spent longer ingesting phloem than emigrants. For emigrants, however, phloem contact was very rarely observed during our recordings. The impact of our findings for understanding the multitrophic interactions between host plant, pathogen and behavior of vector insects are discussed with regard to the epidemiology of AP and pest control strategies of the vector.

The identification and molecular characteristics of Belarusian apple proliferation phytoplasma isolates

It is known that optimal source of samples for diagnostic of apple proliferation phytoplasma in autumn is roots. In case of the occurrence of pronounced characteristic symptoms - “witches’ broom”, can be used symptomatic shoots. Real-time PCR with primer pair Phyto-F/Phyto-R and probe Phyto-P and nested PCR with primer pair P1/Tint and fO1/ rO1 can be used to detect Candidatus Phytoplasma mali with a high degree of sensitivity and reproducibility. Comparison of the nucleotide sequences of Belarusian isolates with the sequences presented in EMBL/GenBank showed that all Belarusian phytoplasma isolates detected on apple cultivars Alesya, Syabryna, Pamyat Sikory belong to species Candidatus Phytoplasma mali. The nucleotide sequences are placed in international database (EMBL/GenBank) with identification numbers (LR701160, LR701188, LR701436, LR701155, LR701438, LR701439, LR701440). The identity of the nucleotide sequences of region of 16S rRNA gene of Belarusian samples of Ca. P. mali ranged from 99.7 to 100.0 %, and hflB gene region ranged from 99.6 to 100.0 %.

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

Simple SummaryApple proliferation disease is one of most important threats for European apple cultivation. It is caused by the bacterium ’ Candidatus Phytoplasma mali’ which is transmitted by the psyllids Cacopsylla picta and Cacopsylla melanoneura in South Tyrol. Since no curative treatment is available against the disease, uprooting of infected trees and phytosanitary measures against the known insect vectors are the only strategies to prevent a spread of the pathogen. Interestingly, densities of the known vector insects and disease manifestation in do not always correlate, indicating that other insect vectors might be involved in the transmissive cycle. To elucidate if other insects are involved in ’ Ca. Phytoplasma mali’ transmission, a faunistic characterization of the Auchenorrhyncha community of the agroecosystem “apple orchard” was performed and potential vectoring insect species were PCR-analyzed to unravel if they carry the pathogen. Several Auchenorrhyncha species were identified that have never been described before in the territory of South Tyrol, but ’ Ca. Phytoplasma mali’ was detected only sporadically in species other than the known vectors. The results provide valuable insights into the Auchenorrhyncha species composition in apple orchards, confirm the prevalent role of the known vectors in the region and are thus an important basis for phytosanitary decision-making. 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.