Candidatus Phytoplasma Prunorum Research Articles

The Effect of Phytoplasma Disease Caused by ‘Candidatus Phytoplasma prunorum’ on the Phenological and Pomological Traits in Apricot Trees

‘Candidatus Phytoplasma prunorum’, the causal agent of European stone fruit yellows (ESFY), is one of the most important pathogens causing considerable economic losses in stone fruit orchards. This study evaluated trees infected and noninfected by phytoplasma ESFY of 16 apricot varieties grown in an orchard in Lednice (Czech Republic) between the years 2008-2014. Pomological traits, phenophases, pollen germination and seed viability were analysed as well as the presence of ‘Ca. P. prunorum’ in pollen, flower organs, fruit flesh, immature seeds and seedlings by nested PCR. One of the most detrimental impacts was the decreased fruit set of infected trees which occurred in 12 out of 16 studied varieties reaching an average fruit set decrease of 16.1%. Pollen germination rates also suffered, showing an average decrease by 11.78% in infected trees. In the analysis of some traits, overall significant differences were observed between the infected and noninfected trees. However, for all analysed traits in different varieties, both positive and negative influences of phytoplasma ESFY were observed. The presence of ‘Ca. P. prunorum’ in infected trees was confirmed in flower parts (only in peduncle in 70.2% of cases) and fruit parts (immature fruit flesh in 42.0% and milky kernels in 26.2% of cases), however, neither in seedlings nor in pollen.

Cage and field experiments as basis for the development of control strategies against Cacopsylla pruni, the vector of European Stone Fruit Yellows

AbstractThe efficacy and the instant effect of 13 insecticides and antifeedants towards Cacopsylla pruni, the vector of ‘Candidatus Phytoplasma prunorum’ were examined in cage studies (no choice experiments with 10 field‐collected overwintered adults per experiment) on potted apricot trees (budding trees under outdoor conditions in early spring and foliated seedlings kept at 21°C). Cypermethrin caused 100% insect mortality within 2–4 h, thiacloprid 90–100% mortality within 24 h both on foliated and on budding trees. On budding trees spinosad led to 70–90% mortality within 24 h, thixotropic white trunk paint to 90% mortality within 48 h. On foliated seedlings flonicamid gave 70–100% mortality within 1 day, abamectin, spinosad, acetamiprid and spirotetramat 70–100% within 72 h. Field studies monitoring the effects of thiacloprid on remigrants of C. pruni by yellow sticky traps were carried out in two apricot orchards. Additionally the influence of the insecticide on insect dispersal was examined by mark, release and recapture trials. As compared to the control thiacloprid significantly reduced the catches of naturally occurring and released insects, decreased the number of trees on which released insects were recaptured (by 25–100%) and shortened the migration distances of the released insects by more than half. Our results suggest that appropriate insecticide treatments both reduce C. pruni populations and have a direct effect on pathogen transmission. Application of Cypermethrin before bloom and thiacloprid after bloom seem best suited to achieve these objectives. Thixotropic white trunk paint could, a formulation for spray application provided, eventually be a sustainable alternative or complement for treatments before bloom.

Transmissible tolerance to European stone fruit yellows (ESFY) in apricot: cross-protection or a plant mediated process?

European Stone Fruit Yellows (ESFY) is an emerging disease caused by ‘Candidatus Phytoplasma prunorum’ (‘Ca. P. prunorum’) affecting stone fruits, as apricots. Resistant apricot cultivars are unknown, but it has been demonstrated that individual plants can recover from the disease, behaving as completely tolerant to ESFY. The status of tolerance is transmissible by grafting to successive apricot individuals, but it is not clear whether recovery corresponds to a transmissible tolerance that depends on a plant–mediated reaction or if it is due to a cross-protection promoted by a transmissible protective agent i.e. hypovirulent strain/s of ‘Ca. P. prunorum’. Results achieved after prolonged field experiments support the first hypothesis. Two groups of apricot plants derived from a common recovered mother (one ‘Ca. P. prunorum’-free after heat-treatment and the second not heat-treated, i.e. harbouring potential protective strain/s of the phytoplasma), behaved similarly: no plants from either of the two groups developed stable ESFY symptoms after natural infections. Corresponding groups of plants, derived from symptomatic mothers, developed a high percentage of diseased plants after natural infection. No potential protective ‘Ca. P. prunorum’ hypovirulent strains were detected in the asymptomatic apricot plants. The summarized evidence supports a host-defence induction, likely of epigenetic feature. The present long-term study in apricot represents an uncommon empiric proof supporting the theory of inducible resistance to pathogens in plants.

A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification of 'Candidatus Phytoplasma prunorum'.

Many efforts have been made to develop a rapid and sensitive method for phytoplasma and virus detection. Taking our cue from previous works, different rapid sample preparation methods have been tested and applied to Candidatus Phytoplasma prunorum (‘Ca. P. prunorum’) detection by RT-qPCR. A duplex RT-qPCR has been optimized using the crude sap as a template to simultaneously amplify a fragment of 16S rRNA of the pathogen and 18S rRNA of the host plant. The specific plant 18S rRNA internal control allows comparison and relative quantification of samples. A comparison between DNA and RNA contribution to qPCR detection is provided, showing higher contribution of the latter. The method presented here has been validated on more than a hundred samples of apricot, plum and peach trees. Since 2013, this method has been successfully applied to monitor ‘Ca. P. prunorum’ infections in field and nursery. A triplex RT-qPCR assay has also been optimized to simultaneously detect ‘Ca. P. prunorum’ and Plum pox virus (PPV) in Prunus.

Incidence and distribution of ‘Candidatus Phytoplasma prunorum’ and its vector Cacopsylla pruni in Spain: an approach to the epidemiology of the disease and the role of wild Prunus

‘Candidatus Phytoplasma prunorum’ is the causal agent of the European stone fruit yellows (ESFY) disease. This phytoplasma affects wild and cultivated species of Prunus to different degrees, depending on their susceptibility. ‘Candidatus Phytoplasma prunorum’ is present in the four regions of Spain surveyed in this study (Aragon, Catalonia, Extremadura and Valencia) with a variable incidence. Results showed that ‘Ca. Phytoplasma prunorum’ was detected in all of the cultivated Prunus species studied, except P. avium and P. dulcis, and was widespread in Spain. The most affected species was P. salicina, with symptoms including early bud break and blooming, leaf curling and yellowing, collapse, and a major decrease in production. In some plots in the Baix Llobregat area of Barcelona province (Catalonia), the incidence of ESFY on P. salicina was as high as 80%. The insect vector, Cacopsylla pruni, was present in all four of the regions studied, with the highest captures in yellow sticky traps in Catalonia on P. mahaleb and in Extremadura in peach orchards. In Baix Llobregat, large populations of C. pruni were present on infected P. mahaleb bushes, and with high infection rates. This was a key factor in the local pathogenic cycle that caused a major ESFY outbreak in the nearby P. salicina orchards. In the Ebro valley (Lleida and Aragon) and Valencia, the surveys showed very low incidences of the disease and low C. pruni populations.

Detection and multigene characterisation of a ‘CandidatusPhytoplasma prunorum’-related phytoplasma associated with witches’ broom symptoms in pineapple fruit

Pineapple samples showing witches’ broom symptoms in the leaves at the top of the fruit were analysed to verify phytoplasma presence. 16SrX group phytoplasmas were detected after nested PCR amplification with specific primers and RFLP analyses. Sequencing of 16Sr DNA allows verification that phytoplasmas from pineapple show a 99% shared identity with ‘Candidatus Phytoplasma prunorum’. Further characterisation of the phytoplasma on ace, imp and secY genes by RFLP analyses on amplicons obtained in nested PCR assays enable the differentiation of the strain infecting pineapple from those detected in fruit trees in Europe and used as reference strains. This is the first report of phytoplasmas belonging to the 16SrX-B group from a species different from pome and stone fruit worldwide.

Molecular diversity of phytoplasmas infecting cherry trees in Poland

Sour cherry with shoot proliferation and dieback symptoms and sweet cherry showing leaf roll and yellowing of the leaves were observed in orchards located in west and central parts of Poland. Results of phylogenetic and RFLP analysis of the partial sequence of 16S rDNA and 16S-23S rDNA spacer region virtually digested with 16 restriction enzymes showed the presence of ‘Candidatus Phytoplasma prunorum’ in sweet cherry and ‘Ca. P. asteris’ in sour cherry trees.

Ideas on the European stone fruit yellows – as an entomologist can see them

The European stone fruit yellows (ESFY) is an important endemic disease in Europe which causes in both, the Mediterranean countries and Central Europe serious damage. Its pathogen is the ‘Candidatus Phytoplasma prunorum’. The treatment and healing of the diseased trees and plantations with chemicals do not promise success. Thus, prevention may be the only solution. The transmission and spread of the pathogen happen by infected propagation material (grafting) or a vector (the psyllid, Cacopsylla pruni). Mechanism of the pathogen’s transmission and population dynamics of the vector have been extensively investigated in several European countries, which may allow by the control of C. pruni even to hold back the disease. Diseased stone fruit trees and wild Prunus spp. as main host species play an important role in maintaining and spreading the pathogen. C. pruni collects the pathogen by feeding on these plants and it carries persistently ‘Ca. P prunorum’. Researchers in Hungary have been characterized the disease only in terms of plant pathology, but neither the significance of the vector nor the role of wild Prunus spp. have been studied. This summary intends to give clues to these researches, that not only axe and saw should be the instruments of national control, but knowing the role and population dynamics of the vector the stone fruit production should be more successful.

Phytoplasma diseases on fruits in Hungary

In the last twenty years, three phytoplasma diseases were identified in Hungary, viz. European Stone Fruit Yellows (ESFY) (caused by Candidatus Phytoplasma prunorum), pear decline (caused by Candidatus Phytoplasma pyri), and apple proliferation (caused by Candidatus Phytoplasma mali). Candidatus Phytoplasma prunorum was isolated from apricot, peach, plum and japanese plum. Cacopsylla pruni the vector of ESFY was also isolated and identified. Infection of Candidatus Phytoplasma pyri was diagnosed from pear and Candidatus Phytoplasma mali was found on apple and pear. The three phytoplasmas cause different damages on their host plants. The most economically important phytoplasma disease is the ESFY. It seriously impairs apricot and japanase plum trees. After infection of apricots and japanese plums show yellowing and defoliation, and within a few years die in apoplexy-like symptoms. The disease on japanese plum is so severe that this fruit practically can not be cultivated in Hungary. Pear decline is the most serious problem especially in intensive pear plantations. The vector Cacopsylla pyri, C. pyrisuga and C. pyricola can be found in almost all pear orchards. Because of the regular presence of psyllids in intensive pear orchards the insecticide control is necessary. Apple proliferation is not an important disease in Hungary. All of our isolations of ’Candidatus Phytoplasma mali’ occured in organic orchards and record was not available in Hungary lately.

First Report of 'Candidatus Phytoplasma pyri' Causing Peach Yellow Leaf Roll (PYLR) in Spain.

'Candidatus Phytoplasma prunorum,' which causes European stone fruit yellows (ESFY), is the prevalent phytoplasma affecting Prunus spp. in Europe. It is closely related to 'Ca. P. pyri,' which causes pear decline (PD) in pear trees. Both phytoplasma belong to the ribosomal group 16Sr-X and are naturally transmitted by different species of Cacopsylla spp. (4). In North America, 'Ca. P. pyri' is responsible for peach yellow leaf roll (PYLR), transmitted by Cacopsylla pyricola from pear to peach trees (1). In Spain, 'Ca. P. prunorum' is widespread on Prunus spp., but its occurrence on Prunus persicae is very low and 'Ca. P. pyri' is present in every pear orchard (3). During 2012, a previously unreported syndrome including early reddening, leaf curling, decline, abnormal fruits, and in some cases chlorosis and death of peach trees was reported on peach in Lleida, northern Spain. Symptoms were different to ESFY and PYLR, in that flowering disorders such as ESFY or yellows were not apparent, and reddening and decline were the most common symptoms. The disease was present in a wide range of varieties and rootstocks, suggesting insect transmission in an area where C. pruni, vector of 'Ca. P. prunorum,' was not previously reported, but C. pyri was abundant in pear orchards. Shoot samples from 20 symptomatic peach trees were collected in seven orchards within a 2 km2 area with an estimated incidence of 40%, which was higher in the borders. DNA was extracted from 1 g of leaf midribs and phloem tissue and amplified with ribosomal universal primers P1/P7 followed by nested PCR with R16F2n/R16R2 and specific primers fO1/rO1 that target the 16Sr-X group (3). The final PCR products were digested with RsaI enzyme. Amplifications with non-ribosomal specific primers, Imp ESFY, Imp PD A and Imp PD B that amplify sequences of gene Imp, that encode a phytoplasma membrane protein, were also carried out (2). Tissue samples with ESFY and PD and peach seedlings were used as positive and negative controls, respectively. Amplified PCR products were sequenced and compared to sequences deposited in GenBank. Phytoplasmas were detected in 18 of the 20 samples analyzed. No phytoplasmas were detected in negative peach controls. All digestions of fO1/rO1 PCR products from peach samples showed a PD profile, while no ESFY profile was detected. All samples were positive with specific primers Imp PD A and B. None of the peach samples were positive with the specific Imp-ESFY primers. Sequencing of R16 and Imp PDA and B amplicons revealed the presence of a stable isolate. The sequences were submitted to the European nucleotide archive (ENA) with the accession nos. HG737345 and HG737344. Based on the 16S rDNA sequence, this strain is 100% homologous to the reference strain PD1 (GenBank Accession No. AJ542543) and 99.55% homologous to strain PD 33 Lib (GenBank FN600725) based on the Imp gene sequence. This is the first report of PD phytoplasma in peach trees in Spain, and the first report in Europe of PD phytoplasma causing economically important outbreaks in peach orchards, following a pattern that could be similar to PYLR in North America. This strain is genetically closer to some European or Middle Eastern PDs than to North American PYLR.

Cross-protection test with ‘CandidatusPhytoplasma mali’ 1/93 strain against closely and distantly related phytoplasma strains

Putative inhibitory effect of ‘Candidatus Phytoplasma mali’ 1/93 mild strain was tested under greenhouse conditions against the related ESFY and the distantly related STOL and AAY strains of ‘Candidatus Phytoplasma prunorum’, ‘Candidatus Phytoplasma solani’ and ‘Candidatus Phytoplasma asteris’, respectively, which are responsible for serious damages and yield losses in fruit orchards across Europe. A consequent decrease of the DNA amount of the virulent GSFY strain analysed by PCR and Real-Time PCR could be observed when the plants were preliminary immunised by 1/93 strain. The cross-protection phenomenon proved to be effective between closely related strains, but no inhibition occurred among distantly related strains due to preliminary infection. Our results support that immunization with a mild, antagonist strain can be of phytopathological relevance in phytoplasmal researches.

Spread of European fruit tree phytoplasma diseases

The genetically closely related ‘Candidatus Phytoplasma mali’, ‘Candidatus Phytoplasma prunorum’ and ‘Candidatus Phytoplasma pyri’ are associated with economically important diseases such as apple proliferation, European stone fruit yellows and pear decline in European fruit tree areas and are spread by psyllid vectors of the superfamily Psylloidea (Hemiptera, Sternorrhyncha). Although the relationship between these phytoplasmas, their hosts and the vectoring psyllids is almost highly specific, there are some interconnections and biological factors which influence disease epidemiology and vector ecology and which should be taken into consideration for risk assessment and vector control. For a reliable risk assessment of a putative vector is important to know its population dynamics, its transmission efficiency, its host plant preferences and its epidemiological cycle. Although psyllid vectors usually show a high host plant specificity, being either monophagous or oligophagous on closely related plant species, shelter plants may serve as sources of inoculum and hence interfere with the disease spread. Therefore, the knowledge of the epidemiological cycle of both, the phytoplasma and its main vector is crucial for the development of efficient control measures.

Distribution of ‘Candidatus Phytoplasma prunorum’ and its vector Cacopsylla pruni in European fruit‐growing areas: a review

European stone fruit yellows (ESFY) is an EU‐listed I/AII disease affecting Prunus spp. caused by ‘Candidatus Phytoplasma prunorum’. This paper reports the results from a systematic literature review approach that sought to determine the geographic distribution of ‘Ca. Phytoplasma prunorum’ in European fruit‐growing areas. Evidence for the presence of the phytoplasma was found for 15 of the 27 EU countries. It is prevalent in the most important stone fruit production areas of Central and Southern Europe, where it causes substantial impact in apricots (Prunus armeniaca), Japanese plums (P. salicina) and peaches (P. persica). In Northern European areas where these hosts are not produced, it is occasionally found on tolerant species (P. domestica). However, because surveys of the disease status of tolerant hosts are not performed, it remains unclear whether the pathogen is absent in Northern Europe or survives in tolerant cultivated or wild hosts. No reports of ESFY were found from the southernmost part of Europe: Portugal, Spain (Andalucia, Castile–La Mancha), Italy (Sicily, Puglia), Greece (Crete), Cyprus and Malta. This may be explained by the absence of the favoured wild hosts of the vector. Moreover, it remains unclear if the vector finds suitable conditions for aestivation and overwintering in these regions.

First report of 'Candidatus Phytoplasma prunorum' infecting apricots in Tunisia.

‘Candidatus Phytoplasma prunorum’ was detected for the first time in Tunisia in apricot trees that showed early leaf reddening in autumn, off-season growth in winter followed by dieback, and bore small and tasteless fruits. Phytoplasma was detected by nested PCR using the universal phytoplasma primer pairs R16mF2/R1 and R16F2n/R2. An amplification product of the expected size (1.2 kbp) was obtained from samples collected from symptomatic but not from symptomless apricot trees. Amplicons subjected to RFLP analysis after digestion with endonucleases MseI and RsaI, gave patterns similar to those reported for the European stone fruit yellows phytoplasma (ESFY, 16SrX-B). Identification was further confirmed by PCR using ESFY-specific primer pairs (ECA1/ECA2).

Less Common Phytoplasmas Infecting Stone Fruit Trees

Less Common Phytoplasmas Infecting Stone Fruit TreesPrunusspecies plants can be infected by eight ‘CandidatusPhytoplasma’ (‘Ca.P.’) species classified to eight distinctive taxonomic groups: ‘Ca.P. prunorum’ (16SrX-B), ‘Ca.P. mali’ (16SrX-A), ‘Ca.P. pyri’ (16SrX-C), ‘Ca.P. asteris’ (16SrI), ‘Ca.P. aurantifolia’ (16SrII), ‘Ca. P. ziziphi’ (16SrV), ‘Ca.P. fraxini’ (16SrVII), ‘Ca.P. phoenicium’ (16SrIX) and two potentially new species: ‘Ca.P. pruni’ (16SrIII) and ‘Ca.P. solani’ (16SrXII). These agents occur incidentally in orchards and their impact on stone fruit production is lower than ‘CandidatusPhytoplasma prunorum’. Hosts, geographic distribution, symptoms and insect vectors of these ‘Ca.P.’ species, methods of their identification, and control management are reviewed.

First report of Candidatus Phytoplasma prunorum infecting almonds in Tunisia

Candidatus Phytoplasma prunorum was detected for the first time in almond (Prunus dulcis Mill.) cv. ‘Abiod’ in Tunisia. Infected trees showed emergence of new growth during dormancy and leafed out before flowers opened in addition to early defoliation in summer. Phytoplasma was detected by nested polymerase chain reaction (PCR) using universal phytoplasma primer pairs P1/P7 and F2n/R2. A band with expected size was observed in samples collected from five symptomatic, but not symptomless almond trees. PCR products (1.2 kbp) were used for restriction fragment length polymorphism (RFLP) analysis after digestion with endonucleases RsaI and SspI. RFLP patterns obtained were similar to those reported previously for the European stone fruit yellows (ESFY, 16SrX-B). Identification has been further confirmed by PCR using ESFY specific primer pairs (ECA1/ECA2). This is the first report of Ca. Phytoplasma prunorum infecting almonds in Tunisia.

Phytoplasma diseases of stone fruit trees in Turkey and their containment

Although fruit tree phytoplasmas were studied since 1999 in Turkey, there have been very limited studies and records on stone fruit tree phytoplasmas. The main symptoms reported on apricot, plum, peach and almond were chlorosis between veins, off season flowering and fruiting as a result of early bud breaking, longitudinal leaf rolling and quick die-back. More than 500 cultivated and wild Prunus plants in or nearby germplasm nurseries and commercial orchards during 2002–2009 were tested by using universal primers P1/P7 and fU5/rU3 in direct and nested PCR assays, respectively. Amplification products were digested with RsaI and SspI restriction enzymes. The average incidence of ‘Candidatus Phytoplasma prunorum’ was detected as 10.19%. The most infected stone fruit species were apricot and plum, followed by almond and peach. No phytoplasma was found in cherries and wild Prunus species.

European Stone Fruit Yellows Disease and Its Causal Agent ‘Candidatus Phytoplasma Prunorum’

The European stone fruit yellows (ESFY) caused by ‘Candidatus Phytoplasma prunorum’ (‘Ca. P. prunorum’), is a devastating, quarantine phytoplasma disease. This agent leads to serious damage in apricot and Japanese plum orchards, including decline of infected trees. The consequences are considerable economic losses in production. European plum and cherry are less susceptible to ESFY, although the ‘Ca. P. prunorum’ was also detected in these trees in several countries. ESFY seems to be limited to Europe and the neighboring regions (Asia Minor) as its incidence was reported from most of the southern and central European countries, Turkey, and Azerbaijan. ‘Ca. P. prunorum’ is transmitted by Cacopsylla pruni (Scopoli). The impact of ‘Ca. P. prunorum’ on stone fruit trees, as well as the methods of detection and identification and control management are reviewed.

Survey on the presence of Cacopsylla pruni in Turkey: preliminary results

Although ‘Candidatus Phytoplasma prunorum’ has been previously detected in different regions of Turkey, its vectors have not been identified yet. A survey was conducted in 2010 and 2011 in order to determine the presence of Cacopsylla pruni populations in six different fruit tree producing areas. The psyllid was found in Adana, Mersin, Bursa and Yalova on either Prunus spinosa or wild plums (Prunus spp.) and also on fir trees. The mean percentage of individuals of C. pruni infected with Ca. P. prunorum was around 23% in Mersin.

Dieback of apricot plantations caused by 'Ca. Phytoplasma prunorum' in Borsod-Abaúj-Zemplén county (Northern-Hungary)

Plant diseases caused by phytoplasmas have increasing importance in all over the world for fruit growers. Lately, phytoplasma diseases occur on many fruit varieties and responsible for serious losses both in quality and quantity of fruit production. In the long-run these diseases cause destruction of fruit trees. The apricot phytoplasma disease (Ca. Phytoplasma prunorum) was first reported in Europe in 1924 from France. In 1992 the disease has also been identified in Hungary. On the base of growers' signals serious damages of "Candidatus Phytoplasma prunorum" Seemüller and Schneider, 2004 (formerly: European stone fruit yellows phytoplasma) could be observed in different stone fruit plantations in the famous apricot-growing area nearby Gönc town, Northern-Hungary. Field examinations have been begun in 2009 in several stone fruit plantations in Borsod-Abaúj-Zemplén County mainly in Gönc region which is one of the most important apricot growing regions in Hungary, named “Gönc Apricot Growing Area”. Our goals were to diagnose the occurrence of Ca. Phytoplasma prunorum on stone fruits (especially on apricot) in the North-Hungarian growing areas by visual diagnostics and confirm data by laboratory PCR-based examinations. All the 28 collected samples were tested in laboratory trials and at 13 samples from apricot, peach, sour cherry and wild plum were confirmed the presence of phytoplasma (ESFY). On the base of observations it seems evident that the notable losses caused by "Ca. Phytoplasma prunorum" is a new plant health problem to manage for fruit growers, especially apricot producers in Hungary.