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•Spectral signatures allow the differentiation of phytoplasma infected apple trees.•Identification of wavelength regions relevant for differentiation.•Sampling date and phytoplasma ...colonization of the canopy impact the method’s success.•Non-destructive detection method for identifying phytoplasma infected apple trees.•On-site Application of this method possible.
'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.
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•The reliability of the AP classification increases throughout vegetation period.•Best reliability of the AP classification coincides with high APP concentrations.•Differentiation was ...possible independently of symptom expression in the leaves. - Spectral regions of carbohydrates and N-compounds were differentially affected.
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.
Phytoplasmas are insect-vectored, difficult-to-culture bacterial pathogens that infect a wide variety of crop and non-crop plants, and are associated with diseases that can lead to significant yield ...losses in agricultural production worldwide. Phytoplasmas are currently grouped in the provisional genus ‘
Candidatus
Phytoplasma’, which includes 49
‘Candidatus’
species. Further differentiation of phytoplasmas into ribosomal groups is based on the restriction fragment length polymorphism (RFLP) pattern of the 16S rRNA-encoding operon, with more than 36 ribosomal groups (16Sr) and over 100 subgroups reported. Since disease symptoms on plants are not associated with phytoplasma identity, accurate diagnostics is of critical importance to manage disease associated with these microorganisms. Phytoplasmas are typically detected from plant and insect tissue using PCR-based methods targeting universal taxonomic markers. Although these methods are relatively sensitive, specific and are widely used, they have limitations, since they provide limited resolution of phytoplasma strains, thus necessitating further assessment of biological properties and delaying implementation of mitigation measures. Moreover, the design of PCR primers that can target multiple loci from phytoplasmas that differ at the sequence level can be a significant challenge. To overcome these limitations, a PCR-independent, multilocus sequence typing (MLST) assay to characterize an array of phytoplasmas was developed. Hybridization probe s targeting
cpn60
,
tuf
,
secA
,
secY
, and
nusA
genes, as well as 16S and
rp
operons, were designed and used to enrich DNA extracts from phytoplasma-infected samples for DNA fragments corresponding to these markers prior to Illumina sequencing. This method was tested using different phytoplasmas including ‘
Ca
. P. asteris’ (16SrI-B), ‘
Ca
. P. pruni’ (16SrIII-A),‘
Ca
. P. prunorum’ (16SrX-B), ‘
Ca
. P. pyri’ (16SrX-C), ‘
Ca
. P. mali’ (16SrX-A), and ‘
Ca
. P. solani’ (16SrXII-A). Thousands of reads were obtained for each gene with multiple overlapping fragments, which were assembled to generate full-length (typically >2 kb), high-quality sequences. Phytoplasma groups and subgroups were accurately determined based on 16S ribosomal RNA and
cpn60
gene sequences. Hybridization-based MLST facilitates the enrichment of target genes of phytoplasmas and allows the simultaneous determination of sequences corresponding to seven different markers. In this proof-of-concept study, hybridization-based MLST was demonstrated to be an efficient way to generate data regarding ‘
Ca
. Phytoplasma’ species/strain differentiation.
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•Phloem and xylem ingestion differ between remigrants, emigrants and nymphs.•All life stages of this phloem feeding insect also feed on xylem.•Nymphs more often ingest phloem and ...xylem than adults.•Phytoplasma infection of the host plant affects probing behavior of the vector.•Phloem salivation occurs later on infected 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.
Summary
The plant pathogen Candidatus Phytoplasma mali (P. mali) is the causative agent of apple proliferation, a disease of increasing importance in apple‐growing areas within Europe. Despite its ...economic importance, little is known about the molecular mechanisms of disease manifestation within apple trees. In this study, we identified two TCP (TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of Malus x domestica as binding partners of the P. mali SAP11‐like effector ATP_00189. Phytohormone analyses revealed an effect of P. mali infection on jasmonates, salicylic acid and abscisic acid levels, showing that P. mali affects phytohormonal levels in apple trees, which is in line with the functions of the effector assumed from its binding to TCP transcription factors. To our knowledge, this is the first characterization of the molecular targets of a P. mali effector and thus provides the basis to better understand symptom development and disease progress during apple proliferation. As SAP11 homologues are found in several Phytoplasma species infecting a broad range of different plants, SAP11‐like proteins seem to be key players in phytoplasmal infection.
Apple proliferation (AP) represents a serious threat to several fruit-growing areas and is responsible for great economic losses. Several studies have highlighted the key role played by the cell wall ...in response to pathogen attack. The existence of a cell wall integrity signaling pathway which senses perturbations in the cell wall architecture upon abiotic/biotic stresses and activates specific defence responses has been widely demonstrated in plants. More recently a role played by cell wall-related genes has also been reported in plants infected by phytoplasmas. With the aim of shedding light on the cell wall response to AP disease in the economically relevant fruit-tree
Malus
×
domestica
Borkh., we investigated the expression of the cellulose
(CesA)
and callose synthase
(CalS)
genes in different organs (i.e., leaves, roots and branch phloem) of healthy and infected symptomatic outdoor-grown trees, sampled over the course of two time points (i.e., spring and autumn 2011), as well as in in vitro micropropagated control and infected plantlets. A strong up-regulation in the expression of cell wall biosynthetic genes was recorded in roots from infected trees. Secondary cell wall
CesAs
showed up-regulation in the phloem tissue from branches of infected plants, while either a down-regulation of some genes or no major changes were observed in the leaves. Micropropagated plantlets also showed an increase in cell wall-related genes and constitute a useful system for a general assessment of gene expression analysis upon phytoplasma infection. Finally, we also report the presence of several ‘knot’-like structures along the roots of infected apple trees and discuss the occurrence of this interesting phenotype in relation to the gene expression results and the modalities of phytoplasma diffusion.
'Candidatus Phytoplasma mali', the causal agent of apple proliferation disease, exerts influence on its host plant through various effector proteins, including SAP11
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 SAP11
. For that purpose, leaves of Nicotiana occidentalis H.-M. Wheeler were Agrobacterium-infiltrated to induce transient expression of SAP11
and changes in the transcriptome were recorded until 5 days post infiltration.
The RNA-seq analysis revealed that presence of SAP11
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.
The results indicate that early SAP11
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.
Infochemicals mediate communication within and between different trophic levels. In this study, we identified a new type of plant allomone induced by a plant pathogen and perceived by its vector ...insect Cacopsylla picta. This phloem-feeding psyllid is the main vector of Candidatus Phytoplasma mali, a cell wall-lacking bacterium that causes the so-called apple proliferation disease. In a previous study, we showed that newly emerged females of C. picta were attracted by the odor of phytoplasma-infected apple plants (Malus domestica), which release ß-caryophyllene in contrast to uninfected plants. Here, the attractiveness of this sesquiterpene for C. picta was confirmed in both olfactometer bioassays and field studies. Synthetic ß-caryophyllene was highly attractive to newly emerged adults of C. picta both when offered simultaneously with healthy apple odor and without. The psyllid's response was independent of its odor experience and infection status. These results confirm our previously established hypothesis that this phytoplasma manipulates the behavior of its vector insect by changing the odor blend of its host plant. Deployed in apple orchards, sticky traps baited with ß-caryophyllene dispensers caught both males and females of C. picta. Consequently, this new type of infochemical, i.e., a phytopathogen-induced plant allomone, represents a promising compound to develop innovative techniques for monitoring or maybe even mass trapping of C. picta.
Cacopsylla picta (Förster, 1848) (Hemiptera: Pysllidae) is the main vector of apple proliferation, a phytoplasma-caused disease. It represents one of the most severe problems in apple orchards, and ...therefore, there is a mandatory requirement to chemically treat against this pest in the European Union. Sexual communication using substrate-borne vibrations was demonstrated in several psyllid species. Here, we report the characteristics of the vibrational signals emitted by C. picta during courtship behavior.The pair formation process can be divided into two main phases: identification and courtship. Females initiate the communication on the host plant by emitting trains of vibrational pulses and, during courtship, if males reply, by emitting a signal consisting of a series of pre-pulses and a ‘buzz’, a duet is established. Moreover, a scanning electron microscopy investigation showed the presence of a stridulatory structure on the thorax and wings of both sexes, whereas the video recordings elucidated associated wing movement.The results provide new information about the biology of this phytoplasma vector and could form a basis of an environmentally friendly pest management strategy.
The phloem‐sucking psyllid Cacopsylla picta plays an important role in transmitting the bacterium ‘Candidatus Phytoplasma mali’, the agent associated with apple proliferation disease. The psyllid can ...ingest ‘Ca. Phytoplasma mali’ from infected apple trees and spread the bacterium by subsequently feeding on uninfected trees. Until now, this has been the most important method of ‘Ca. Phytoplasma mali’ transmission. The aim of this study was to investigate whether infected C. picta are able to transmit ‘Ca. Phytoplasma mali’ directly to their progeny. This method of transmission would allow the bacteria to bypass a time‐consuming reproductive cycle in the host plant. Furthermore, this would cause a high number of infected F1 individuals in the vector population. To address this question, eggs, nymphs and adults derived from infected overwintering adults of C. picta were reared on non‐infected apple saplings and subsequently tested for the presence of ‘Ca. Phytoplasma mali’. In this study it was shown for the first time that infected C. picta individuals transmit ‘Ca. Phytoplasma mali’ to their eggs, nymphs and F1 adults, thus providing the basis for a more detailed understanding of ‘Ca. Phytoplasma mali’ transmission by C. picta.
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