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.
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 (
), which can be infected by '
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
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
and
mesophyll protoplasts, translocation of both PME2 variants in the cell nucleus was observed. Overexpression of the effector protein affected cell integrity in
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.
In continuing previous work on the role of AAA+ proteins of the apple proliferation agent ‘
Candidatus
Phytoplasma mali’ in phytoplasma virulence and suppression of virulence, 147 full-length deduced ...protein sequences of AAA+ ATPase AP460 of single- and multiple-strain accessions were examined. This approach revealed that in this protein two regions can be distinguished. Region 1, located in the N-terminal part, is characterized by 14 highly conserved substitutions associated with the suppression of virulence. However, these substitutions were not present in all attenuated strains. In the more diverse region 2, located in the C-terminal part, highly conserved substitutions associated with two groups of virulence were identified. In addition to the virulence-related residues, three other groups of conserved substitutions are present in strains with attenuated virulence with or without the presence of suppression-associated substitutions in region 1. In one of these groups, substitutions next to key residues of the ATPase motifs sensor 1 and 2 and arginine finger do occur that seem to affect ATPase function. This group of substitution was present in all attenuated strains. From these findings it can be concluded that two different mechanisms of suppression exist of which the supposed effect on ATPase function seems to be more important than the suppression-associated substitutions in region 1. The presence of virulent, avirulent and suppressive strains in a tree leads in the commonly occurring multiple infections to interactions in which the resulting virulence is determined by the numerical relation of virulent and attenuated strains.
Phytoplasmas are pathogenic bacteria within the class of Mollicutes, which are associated with more than 1000 plant diseases. In this study, we applied quantitative mass spectrometry to analyse ...affected pathways of the model plant tobacco (Nicotiana occidentalis) upon Candidatus Phytoplasma mali strain AT infection. Using tissue obtained from leaf midribs, 1466 plant‐assigned proteins were identified. For 1019 of these proteins, we could reproducibly quantify the expression changes of infected versus noninfected plants, of which 157 proteins were up‐ and 173 proteins were downregulated. Differential expression took place in a number of pathways, among others strong downregulation of porphyrin and chlorophyll metabolism and upregulation of alpha‐linolenic acid metabolism, which was consistent with observed increased levels of jasmonic acid, a key signal molecule of plant defence. Our data shed light on the molecular networks that are involved in defence of plants against phytoplasma infection and provide a resource for further studies.
The mosaic leafhopper,
(Matsumura), is an Asian species widespread in Europe that can cause leaf damage in wild trees and transmit disease phytoplasmas to grapevines. Following an
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
life cycle, leaf symptoms associated to its trophic activity, and its capability to acquire "
Phytoplasma mali," a causal agent of Apple Proliferation (AP). The results indicate that
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
has the potential to be a new apple tree pest. However, further studies are required to better understand the economic impact of the infestations.
Phytoplasmas from the 16Sr-X apple proliferation (AP) group are quarantine species in Europe and causal agents of the most important diseases of fruit trees within the family
Rosaceae
, namely apple ...proliferation, European stone fruit yellows and pear decline. In this study, a detailed insight into the molecular diversity of isolates of two phytoplasmas from the AP group, i. e. ‘
Candidatus
Phytoplasma mali’ and ‘
Ca
. P. prunorum’ obtained from different orchards in Slovenia, was estimated by a multilocus sequence typing, based on analysis of the genomic regions of
aceF
,
pnp
,
secY
and
imp
. With seven and five genotypes defined for ‘
Ca
. P. mali’ and
‘Ca
. P. prunorum’ isolates, respectively,
imp
was the most variable among the applied markers. On the other hand,
pnp
was the least variable with three genotypes defined for
‘Ca
. P. mali’ isolates and only one for
‘Ca
. P. prunorum’ isolates. The presented results complete the survey of the AP group phytoplasma diversity in Slovenia, which has started with the recent analysis of the ‘
Ca.
P. pyri’. The comparison of results with those from several European countries shows an important genetic diversity of the Slovenian genotypes with some previously unknown. The genotype distribution reflects the geographic position of Slovenia. Additional grafting experiments with apricot trees tolerant to ‘
Ca
. P. prunorum’ demonstrated that the tolerance status is transmissible. Some possible mechanisms involved in the process are discussed.
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.
Phytoplasmas, microorganisms associated with severe plant diseases, are obligate parasites transmitted by phloem-feeding insects.
Cacopsylla melanoneura
and
Cacopsylla picta
are involved in the ...transmission of ‘
Candidatus
Phytoplasma mali,’ the etiological agent of apple proliferation (AP) disease. Research conducted in different geographic regions showed different transmission efficiencies for the two psyllids. In this study, acquisition and transmission trials were carried out to investigate the role of different life stages of these vectors in the epidemiology of AP after a sudden outbreak in northeastern Italy. Both species resulted able to acquire ‘
Ca
. P. mali,’ with higher infection rates recorded in
C. picta
. F1 generations showed a higher acquisition ability compared to adults in both species. ‘
Ca
. P. mali’ transmission was successful: Up to 1.5% of test plants were infected in trials with
C. melanoneura
and up to 10.2% in trials with
C. picta
. Overwintered adults of
C. melanoneura
showed a lower vectoring ability compared to
C. picta
. F1 nymphs and F1 adults, developed on infected plants, resulted in being competent vectors. Data on phytoplasma acquisition suggest a different relationship of ‘
Ca.
P. mali’ with the two species, evidencing a stronger affinity with
C. picta
. Moreover, taking into account the different factors influencing AP transmission, the probability of infection is mainly influenced by the presence of F1 nymphs and the phytoplasma load. In conclusion, this study evidences that
C. picta
is the most effective AP vector in the studied area, even if
C. melanoneura
can represent a potential risk in the presence of high inoculum sources.
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.
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