Apple proliferation is an economically important disease and a threat for commercial apple cultivation. The causative pathogen, the bacterium '
Phytoplasma mali', is mainly transmitted by
, a ...phloem-feeding insect that develops on the apple tree (
spp.). To investigate the feeding behavior of adults of the phytoplasma vector
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
and
. DNA from 23 plant genera (
) and four plant families (
) 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
.
Over the years, real-time PCR outflanked endpoint PCR in phytopathogen diagnostics, mainly because of the increase in sensitivity and timesaving aspects of the technique. However, a time consuming ...16S rRNA-based nested PCR method is still the gold standard for phytoplasma diagnosis. This is also the case for phytoplasma detection in
Malus
,
Pyrus
and
Prunus
, the three main host plants of apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) phytoplasma, respectively. The last decade, loop-mediated isothermal amplification (LAMP) (Notomi et al.
2000
) is gaining a lot in significance and is also for phytoplasmas expected to become a widely used reliable diagnostic tool. High specificity and sensitivity which also requires a less stringent need for DNA purification, and the short analysis time and the limited equipment requirements makes the LAMP method a fast and affordable alternative with great point-of-care diagnostic potential. In this paper, we present a LAMP primer set for the ribosomal group 16SrX, containing the important fruit tree phytoplasmas AP, PD and ESFY. The primers were developed and validated for fast and sensitive detection and general use for diagnosis. We foresee that the LAMP technique will also have its application in on-site diagnosis of the fruit tree phytoplasmas during inspections and surveys.
Many phytopathogens that cause worldwide losses of agricultural yield are vectored by herbivorous insects. Limited information is available about the interactions among phytopathogens, host plants, ...and insect vectors. In this paper, we report that the cell wall-lacking bacterium
Candidatus
Phytoplasma mali can alter both the odor of its host plant (apple) and behavior of its vector, the univoltine psyllid
Cacopsylla picta.
Apple trees infected by this phytoplasma emitted higher amounts of β-caryophyllene when compared to uninfected ones. Psyllids that had no previous contact with
Ca.
P. mali, as well as infected pyllids, are more attracted by volatiles emitted from phytoplasma-infected apple plants than from uninfected ones. Psyllids that had developed on infected plants without getting infected showed the opposite behavior. These results suggest that the pathogen modifies host plant odor that lures its vector to infected plants. This may result in higher numbers of transmitting vector insects within the population.
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 '
Phytoplasma mali' load in
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
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.
Detailed information on plant penetration activities by pear psylla Cacopsylla pyri L. (Hemiptera Psyllidae) is essential to study phytoplasma transmission of "Candidatus Phytoplasma pyr" responsible ...of pear decline disease (PD) and to trace and evaluate resistant traits in new pear tree selections for advanced breeding programs. The electrical penetration graph technique or (full) EPG may relevantly contribute to this knowledge. C. pyri EPG waveforms were characterized on basis of amplitude, frequency, voltage level, and electrical origin. Additionally, stylet tracks and the putative location of stylet tips in the plant tissue were histologically related to EPG waveforms by light and transmission electron microscopy observations after stylectomy. More than one waveform occurred in the same tissue: PA, PB, PC1 and PC2 were all detected in the mesophyll, and PE1 and PE2 were both recorded in the phloem. Waveform PE1 was always preceded by transient waveform PD, as previously described in other psyllids. Interestingly, no brief intracellular punctures (potential drop waveforms) were observed during plant penetration, opposite of what is usually recorded in aphids and other Sternorrhyncha.
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 '
Phytoplasma mali', which is ...transmitted by
(Foerster) and
(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
Kopp and Yonk (Hemiptera: Membracidae). However, the concentration of phytoplasma was much lower than in infected
and
captured in the same region, confirming the role of the latter mentioned psyllids as the main insect vectors of AP- phytoplasma in South Tyrol.
1
We elucidated the life cycles of two jumping plant lice species (Hemiptera: Psyllidae): Cacopsylla picta, a vector of the apple proliferation phytoplasma (Candidatus Phytoplasma mali), and ...Cacopsylla melanoneura, a nonvectoring species in Germany and some neighbouring countries, which may transmit the phytoplasma in one region in Italy.
2
The adults of C. picta reproduce exclusively on apple and migrate soon after emergence (emigrants) to conifers in mountainous regions, and return to apple plants in early spring (remigrants). Cacopsylla melanoneura also uses conifers as overwintering host plants but prefers to reproduce on hawthorn, despite its ability to reproduce on apple.
3
Both psyllid species used chemical cues for the identification of their alternate host plants during migration. Remigrants of C. melanoneura preferred the odour of their main reproduction host plant hawthorn to apple but preferred the odour of apple when experienced by feeding and oviposition. Although emigrants of C. picta reportedly prefer the odour of apple trees infected by Ca. P. mali, the remigrants of both species did not distinguish between the odours of infected or uninfected apple plants.
4
Investigating the distribution of Ca. P. mali in plant species involved in psyllid life cycle revealed that the phytoplasma is specialized on apple.
5
Infection of apple by Ca. P. mali increased mortality and resulted in decreased body size of C. picta offspring.
6
Gravid females of C. picta preferred to oviposit on non‐infected plants.
7
It is concluded that Ca. P. mali indirectly promotes its acquisition from infected plants and transmission to non‐infected plants by behavioural manipulation of its vector C. picta.
‘
Candidatus
Phytoplasma mali’, the causal agent of apple proliferation (AP) disease, is a quarantine pathogen controlled by chemical treatments against insect vectors and eradication of diseased ...plants. In accordance with the European Community guidelines, novel strategies should be developed for sustainable management of plant diseases by using resistance inducers (e.g. endophytes). A basic point for the success of this approach is the study of endophytic bacteria associated with plants. In the present work, endophytic bacteria living in healthy and ‘
Ca.
Phytoplasma mali’-infected apple trees were described by cultivation-dependent and independent methods. 16S rDNA sequence analysis showed the presence of the groups
Proteobacteria
,
Acidobacteria
,
Bacteroidetes
,
Actinobacteria
,
Chlamydiae
, and
Firmicutes
. In detail, library analyses underscored 24 and 17 operational taxonomic units (OTUs) in healthy and infected roots, respectively, with a dominance of
Betaproteobacteria
. Moreover, differences in OTUs number and in CFU/g suggested that phytoplasmas could modify the composition of endophytic bacterial communities associated with infected plants. Intriguingly, the combination of culturing methods and cloning analysis allowed the identification of endophytic bacteria (e.g.
Bacillus
,
Pseudomonas
, and
Burkholderia
) that have been reported as biocontrol agents. Future research will investigate the capability of these bacteria to control ‘
Ca
. Phytoplasma mali’ in order to develop sustainable approaches for managing AP.
The psyllid species Cacopsylla melanoneura (Förster) and Cacopsylla picta (Förster) are vectors of ‘Candidatus Phytoplasma mali’, the causal agent of apple proliferation, one of the economically most ...important apple diseases in Europe. Both vectors are present in apple orchards of South Tyrol and Trentino provinces in Northern Italy. As no direct treatment of the disease is possible, monitoring of the psyllids provides information about the vector presence in the orchards and enables targeted control. Thus, fast and reliable identification of the various psyllids occurring in the apple orchards is required. Morphological differentiation is problematic due to extensive resemblance of some psyllid species especially among females and is error-prone for nymphs. Here we present a rapid and cost-effective polymerase chain reaction-restriction fragment length polymorphism method based on the cytochrome c oxidase subunit I region for the molecular identification of the vector species as well as eight further Cacopsylla species present in the orchards. This method was verified through 98.9% consensus with morphologically identified males, through sequencing and subsequent phylogenetic analysis. In case of doubtful morphological identification of females, the method was able to provide a refined species assignment and could also remarkably facilitate the identification of nymphs.
During 2010-2013, samples from 267 apple trees growing in six regions of Poland were tested for phytoplasma presence. 'Candidatus Phytoplasma mali' was detected in 17 samples from phloem tissue of ...apple shoots showing typical apple proliferation symptoms. Molecular characterization of these strains was conducted using PCR-RFLP and sequence analyses of three regions: ribosomal DNA, ribosomal protein and non-ribosomal DNA fragment including nitroreductase and rhodanese-like protein genes. Fragments of 16S rDNA plus 16S-23S spacer region from the 'Ca. P. mali' strains showed two restriction profiles: P-I and P-II when digested with HpaII enzyme. Moreover, based on results of nitroreductase and rhodanese-like protein genes, these phytoplasmas were grouped into two subtypes: AP-15 and AT-1. The majority of the 'Ca. P. mali' strains was classified to the AP-15 subtype. Two strains were grouped to the AT-1 subtype and another showed both AP-15 and AT-1 profiles. Based on results of analysis of rpl22 and rps3 ribosomal protein genes, most of the 'Ca. P. mali' strains were identified as belonging to rpX-A. Only one strain was affiliated to rpX-B subgroup.
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