Establishment of a quantitative real-time PCR assay for the specific quantification of Ca. Phytoplasma prunorum in plants and insects

A real-time PCR assay for the quantification of Ca. Phytoplasma prunorum has been established which combines the specificity of detection with a low cost method of quantitative PCR. The assay uses the specific primers ECA1/ECA2 with a SYBR Green I protocol. A gene fragment of Ca. P. prunorum with the target of the primers has been cloned and is used as standard for quantification by the standard curve method. The assay has been successfully applied to measure the concentration of Ca. P. prunorum in insects as well as in different kinds of plant samples. Keywords: European stone fruit yellows, Cacopsylla pruni, resistance screenin

Analysis of the acquisition and multiplication efficiency of different strains of Ca. Phytoplasma mali by the vector Cacopsylla picta

Based on previous observations during long-term acquisition and transmission trials, studies were carried out under standardized conditions in order to analyse the acquisition and multiplication efficiencies of different strains of Candidatus Phytoplasma mali by different developmental stages of Cacopsylla picta. The acquisition of Ca. P. mali from micropropagated plants infected with different strains was tested for nymphs, larval stages and new adults of C. picta. When born on infected plants a nearly 100% acquisition was achieved for all strains of Ca. P. mali by C. picta. Differences in acquisition efficiency were observed for new generation adults which acquired the phytoplasma as imagines. The multiplication efficiency of the different Ca. P. mali strains inside the insects was analysed by quantitative real-time PCR. Significant differences in the capacity of the different strains to colonise the insect were found. Despite high acquisition rates only few subsequent transmission events to healthy test plants could be recorded

In vitro screening of interspecific hybrids (Malus spp.) for resistance to apple proliferation

A breeding programme was set up six years ago in Trentino as part of the Project “Scopazzi del Melo - Apple Proliferation” (SMAP) in order to obtain AP resistant apple rootstocks.Twenty-six hybrids generated from the crossings (Malus sieboldii, second generation, x Malus domestica) were micropropagated and studied in standardised conditions. An in vitro screening system for AP resistance previously set for the parents of the crosses was adopted and modified. Specific symptoms of the disease, as well as height and basal proliferation of the shoot and size of the leaves, were recorded in vitro at 3 months post-inoculation. At the same time, phytoplasma concentration was determined in the whole shoot by quantitative RT-PCR. An in vitro disease index taking into account all the above-mentioned parameters was developed.Each healthy genotype was graft-inoculated in triplicate with two different phytoplasma strains after plant rooting and acclimatisation. Phenotype and phytoplasma titre were evaluated in the roots the year after infection.Preliminary results indicated that the resistance trait segregates in the progenies. The resistant genotypes had lower phytoplasma concentrations than the susceptible controls, did not show AP-specific symptoms and their growth was not affected by infection. By comparing the resistant behaviour of the same genotypes, the in vitro screening allows for a quick selection of genotypes that are worth evaluating in the field for agronomic traits.Keywords: Apple Proliferation, Malus sieboldii, resistance screening, quantitative real-time PCR, disease inde

Influence of Apple stem grooving virus on Malus sieboldii-derived apple proliferation resistant rootstocks

Apple stem grooving virus (ASGV, Capillovirus) is widely spread in apple growing regions. As it causes no symptoms on most cultivated apple varieties and rootstocks it is considered latent in Malus x domestica. In Asia, however, ASGV has been found associated with topworking disease of apple rootstocks originating from Malus sieboldii. Recently, M. sieboldii and its hybrids have gained new interest in Europe as they confer resistance to apple proliferation (AP) disease. A new breeding program aiming to develop AP-resistant rootstocks of agronomic value for modern apple culture, reported unexpected tree decline which was to be associated with ASGV. As little information is available on the variability of ASGV isolates in Germany, the complete genome of a German isolate of ASGV associated with tree decline was cloned and sequenced. Sequence comparisons with available ASGV isolates revealed two regions of high variability in the genome. The genetic variability of additional isolates from Germany and other countries were collected and the variable areas characterised. In addition ASGV was successfully maintained in micropropagated apple trees and could be transmitted by in vitro grafting to various genotypes, making it possible to study in vitro the effect of the virus and virus/phytoplama combination on M. sieboldii-derived genotypes. Keywords: Latent apple viruses, Candidatus Phytoplasma mali, micropropagation, in vitro grafting, genetic variabilit

Development Of A New Probe For Specific And Sensitive Detection Of 'Candidatus Phytoplasma Mali' In Inoculated Apple Trees

peer reviewe

Reference standardization and triglyceride interference of a new homogeneous HDL-cholesterol assay compared with a former chemical precipitation assay

A homogeneous HDL-c assay (HDL-H), which uses polyethylene glycol-modified enzymes and sulfated alpha-cyclodextrin, was assessed for precision, accuracy, and cholesterol and triglyceride interference. In addition, its analytical performance was compared with that of a phosphotungstic acid (PTA)/MgCl2 precipitation method (HDL-P). Within-run CVs were < or = 1.87%; total CVs were < or = 3.08%. Accuracy was evaluated in fresh normotriglyceridemic sera using the Designated Comparison Method (HDL-H = 1.037 Designated Comparison Method + 4 mg/L; n = 63) and in moderately hypertriglyceridemic sera by using the Reference Method (HDL-H = 1.068 Reference Method - 17 mg/L; n = 41). Mean biases were 4.5% and 2.2%, respectively. In hypertriglyceridemic sera (n = 85), HDL-H concentrations were increasingly positively biased with increasing triglyceride concentrations. The method comparison between HDL-H and HDL-P yielded the following equation: HDL-H = 1.037 HDL-P + 15 mg/L; n = 478. We conclude that HDL-H amply meets the 1998 NCEP recommendations for total error; its precision is superior compared with that of HDL-P, and its average bias remains below +/-5% as long as triglyceride concentrations are < or = 10 g/L and in case of moderate hypercholesterolemia

Survey of Leafhopper Species in Almond Orchards Infected with Almond Witches'-Broom Phytoplasma in Lebanon

Leafhoppers (Hemiptera: Auchenorrhyncha: Cicadellidae) account for more than 80% of all “Auchenorrhynchous” vectors that transmit phytoplasmas. The leafhopper populations in two almond witches'-broom phytoplasma (AlmWB) infected sites: Tanboureet (south of Lebanon) and Bourj El Yahoudieh (north of Lebanon) were surveyed using yellow sticky traps. The survey revealed that the most abundant species was Asymmetrasca decedens, which represented 82.4% of all the leafhoppers sampled. Potential phytoplasma vectors in members of the subfamilies Aphrodinae, Deltocephalinae, and Megophthalminae were present in very low numbers including: Aphrodes makarovi, Cicadulina bipunctella, Euscelidius mundus, Fieberiella macchiae, Allygus theryi, Circulifer haematoceps, Neoaliturus transversalis, and Megophthalmus scabripennis. Allygus theryi (Horváth) (Deltocephalinae) was reported for the first time in Lebanon. Nested PCR analysis and sequencing showed that Asymmetrasca decedens, Empoasca decipiens, Fieberiella macchiae, Euscelidius mundus, Thamnottetix seclusis, Balclutha sp., Lylatina inexpectata, Allygus sp., and Annoplotettix danutae were nine potential carriers of AlmWB phytoplasma. Although the detection of phytoplasmas in an insect does not prove a definite vector relationship, the technique is useful in narrowing the search for potential vectors. The importance of this information for management of AlmWB is discussed