To improve knowledge about plant/phytoplasma interactions and, in particular, about the ‘recovery’ phenomenon in previously-infected plants, we investigated and compared expression levels of several ...defence-related genes (four pathogenesis-related proteins and three jasmonate-pathway marker enzymes) in apple plants showing different states of health: vigorous (healthy), phytoplasma-infected, and recovered. Real Time-PCR analyses demonstrated that genes are differentially expressed in apple leaf tissue according to the plants’ state of health.
Malus domestica
Pathogenesis-Related protein (
MdPR) 1
,
MdPR 2
and
MdPR 5
were significantly induced in leaves of diseased and symptomatic plants compared to leaves of those plants that were healthy or recovered. On the other hand, levels of all the jasmonate (JA)-pathway marker genes that we selected for this study, were up-regulated in the leaves of recovered plants compared to the diseased ones. In conclusion, our study demonstrated that two different sets of defence genes are involved in the interactions between apple plants and ‘
Candidatus
Phytoplasma mali’ (‘
Ca
. P. mali’) and that these genes are differentially expressed during phytoplasma infection or recovery.
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.
Recovery of apple trees from apple proliferation was studied by combining ultrastructural, cytochemical, and gene expression analyses to possibly reveal changes linked to recovery-associated ...resistance. When compared with either healthy or visibly diseased plants, recovered apple trees showed abnormal callose and phloem-protein accumulation in their leaf phloem. Although cytochemical localization detected Ca2+ ions in the phloem of all the three plant groups, Ca2+ concentration was remarkably higher in the phloem cytosol of recovered trees. The expression patterns of five genes encoding callose synthase and of four genes encoding phloem proteins were analyzed by quantitative real-time reverse transcription-polymerase chain reaction. In comparison to both healthy and diseased plants, four of the above nine genes were remarkably up-regulated in recovered trees. As in infected apple trees, phytoplasma disappear from the crown during winter, but persist in the roots, and it is suggested that callose synthesis/deposition and phloem-protein plugging of the sieve tubes would form physical barriers preventing the recolonization of the crown during the following spring. Since callose deposition and phloem-protein aggregation are both Ca2+-dependent processes, the present results suggest that an inward flux of Ca2+ across the phloem plasma membrane could act as a signal for activating defense reactions leading to recovery in phytoplasma-infected apple trees.
Aims: We investigated the ultrastructural and molecular interactions between ‘Candidatus Phytoplasma mali' and the apple endophyte Epicoccum nigrum in the experimental host Catharanthus roseus to ...determine whether inoculation of endophyte could trigger defence reactions in the host. Methods and Results: Apple proliferation (AP) symptom severity was evaluated in AP-grafted plants that were treated by E. nigrum and compared with untreated controls. Phytoplasma concentration was quantified by real-time PCR in treated and untreated plants. Ultrastructural observations revealed that in endophyte-treated periwinkles, modifications to phytoplasmas, such as irregular shape and cytoplasm confined to the periphery of the cell, and plant cytological changes, such as abundant callose depositions and P-protein aggregations in the sieve elements, occurred. AP-grafted plants that were treated by the endophyte (E. nigrum) showed a reduction in symptom severity; in particular, flowers appeared normal in shape and size, when compared with uninfected controls. Real-time PCR indicated that phytoplasma concentration in AP-grafted plants treated with E. nigrum was about 2·8 times lower than that in untreated ones. Conclusions: These results demonstrated that the inoculation with E. nigrum influenced phytoplasma infection in C. roseus plants; plant ultrastructural modifications allowed us to hypothesize an enhancing host defence response. Significance and Impact of the Study: At present, curative protocols against this phytoplasma are not available. Alternative approaches are thus required to reduce disease spread. Our study might represent a first step in the clarification of plant-phytoplasma-endophyte relationships to find possible strategies for the control of phytoplasma diseases.
In several European countries apple trees are affected by apple proliferation disease, which is usually associated with the presence of ‘Candidatus Phytoplasma mali’. During 2010, samples from ...several apple trees displaying proliferation symptoms were collected throughout the Czech Republic to verify identity of phytoplasmas detected in association with the disease. The majority of the 74 apple trees examined using molecular tools were positive for ‘Ca. P. mali’ presence. The 16S–23S ribosomal genes, the ribosomal protein genes and the nitroreductase and rhodonase like genes were then studied to verify phytoplasma strain variability on multigenic bases. Two RFLP profiles and correspondingly two genetic lineages were found in the PCR-amplified fragments covering the 16S–23S rDNA spacer region. ‘Ca. P. mali’ strains belonging to rpX-A subgroup were identified in the majority of the apple tree sampled, whereas phytoplasmas belonging to the rpX-B subgroup were distributed sporadically. The apple proliferation subtypes AP-15 and AT-2 exhibited nearly equal occurrence; the AT-1 subtype and a mixture of the two or all three of the AP subtypes were infrequently found. The PCR/RFLP results were confirmed by nucleotide sequence analyses of selected ‘Ca. P. mali’ strains.
Analysis of pathological and molecular data of 'Candidatus Phytoplasma mali' accessions from 27 apple trees differing considerably in symptomatology was used to molecularly characterize and classify ...strains of the infecting apple proliferation phytoplasma. Single-strand conformation polymorphism and sequence analysis of a variable fragment of ATP00464-type hflB gene revealed that these sources consisted of single-strain and multiple-strain accessions that occurred in similar numbers. The latter group was composed of two to five distinct strains. Analysis of cloned sequences of mild and severe single-strain accessions resulted in two groups of reads that clustered, according to their virulence, distantly in the phylogram. Based on this data, the clustering patterns of multiple-strain accession sequences indicated that nearly all of them were composed of mild and severe strains. The distinct clustering of sequences representing mild and severe strains was associated with a range of molecular markers at the nucleotide and amino acid level. Data indicate that the virulence of multiple-strain accessions is determined by the ratio of the occurring mild and severe strains in that mild accessions were characterized by the predominance of sequences representing mild strains and vice versa. There is evidence that shifts in the population and other events may occur that drastically alter virulence of multiple-strain accessions.
Apple proliferation (AP), caused by ‘Candidatus Phytoplasma mali', is an economically important disease affecting many apple-growing areas in Europe. A new TaqMan real-time PCR assay was established ...for absolute quantification of ‘Ca. P. mali' by using a single-copy gene of the host plant as a reference, which is amplified with the pathogen DNA in a single-tube reaction. Normalised estimates of phytoplasma concentration are ultimately expressed as the number of phytoplasma cells per host plant cell. The assay was used to monitor the ‘Ca. P. mali' titre over the course of two growing seasons in roots and branches of symptomatic and asymptomatic but AP-positive apple trees. All 252 root samples from symptomatic and asymptomatic trees tested positive, with an average number of 59.8 ± 5.68 (standard error) and 55.1 ± 9.83 ‘Ca. P. mali' per host cell, respectively. From the 378 shoot samples analysed, 81% of the symptomatic and only 20% of the asymptomatic samples were AP-positive with an average number of 9.4 ± 1.04 and 0.7 ± 0.13 ‘Ca. P. mali' per host cell, respectively. This strengthens evidence that not the pathogen occurrence alone but the presence of a certain quantity of ‘Ca. P. mali' in the aerial tree sections is involved in symptom expression. In addition, pronounced seasonality of the phytoplasma concentration was found, not only in branches, but also for the first time in roots of symptomatic and asymptomatic apple trees. Highest phytoplasma levels in roots were detected from December to May.
Psyllids are vectors of phytoplasma, which cause important diseases of pome fruit trees. Psyllid species reported as phytoplasma vectors were captured during 2010–2011 in several pome fruit growing ...regions in Turkey. Potential psyllid vectors of ‘Candidatus Phytoplasma mali’ were collected from Malus spp. (apple), Cydonia oblonga (quince), Crataegus spp. (hawthorn) and also from the overwintering hosts, whereas those of ‘Candidatus Phytoplasma pyri’ were collected from wild and cultured forms of Pyrus spp. (pear) trees. The psyllids were identified morphologically as Cacopsylla picta, C. melanoneura-C. affinis complex, C. crataegi, C. pyrisuga, C. pyri, C. pyricola and other Cacopsylla species. The highestnatural phytoplasma infection rate was found in C. picta followed by C. pyri, C. melanoneura-C. affinis complex and C. crataegi with rates of 4.36, 3.84, 2.77 and 1.67%, respectively. No phytoplasma were detected in C. pyrisuga, C. pyricola, or the other Cacopsylla spp. ‘Ca. P. mali’ was detected in C. picta, C. melanoneura-C. affinis complex and C. pyri; ‘Ca. P. pyri’ was detected in C. picta, C. crataegi, C. melanoneura-C. affinis complex and C. pyri individuals. To our knowledge, this is the first report on the possible psyllid vectors of ‘Ca. P. mali’ in Turkey.
Cacopsylla melanoneura is a univoltine psyllid vector of ‘
Candidatus Phytoplasma mali’, the etiological agent of apple proliferation (AP), a severe disease in European apple orchards. The influence ...of ‘
Ca. P. mali’ on the fitness of
C. melanoneura was studied. In the spring of 2007, male–female pairs of field-collected adults were exposed to ‘
Ca. P. mali’-infected or healthy ‘Golden Delicious’ apple shoots. Exposure to these diseased shoots did not affect the life span of the adult psyllids. However, significantly fewer eggs were laid on the diseased shoots. Furthermore, fewer of the eggs that were laid on the infected plants hatched. Data suggest a detrimental effect of AP phytoplasma on the fitness of
C. melanoneura.
‘Candidatus Phytoplasma mali’ (Ca. P. mali) is the disease agent causing apple proliferation (AP), which has detrimental effects on production in many apple growing areas of Central and Southern ...Europe. The present study investigated transcriptional and biochemical changes related to the sugar metabolism as well as expression of pathogenesis-related (PR) protein genes in in vitro micro-propagated AP-infected and healthy control plantlets with the aim of shedding light on host plant response to ‘Ca. P. mali’ infection. Expression changes between infected and control plantlets were detected by quantitative real-time PCR analysis. The most significant transcriptional changes were observed for genes coding for pathogenesis-related proteins and for heat shock protein 70, as well as for some genes related to the sugar metabolism, such as a sorbitol transporter (SOT5), hexokinase, sucrose-phosphate synthase or granule bound starch synthase. Furthermore, biochemical analyses revealed that infected plantlets were characterized by a significant accumulation of starch and sucrose, while hexoses, such as glucose and fructose, and sorbitol were present at lower concentrations. In summary, the present analysis provides an overview of a gene set that is involved in response to phytoplasma infection and, therefore, it may help for a better understanding of the molecular mechanisms involved in phytoplasma–host plant interaction in apple.
•‘Candidatus Phytoplasma mali’ causes apple proliferation (AP) disease.•Transcriptional profiles were analyzed in in vitro micro-propagated AP-infected.•Pathogenesis-related genes and heat shock protein 70 were examined.•Genes related to sugar metabolism and transports were affected in infected tissues.•Biochemical analyses of sugar contents corroborate transcriptional changes.
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