Scientific Papers Series B Horticulture

AP monitoring performed in Romanian appleorchards, based on morphologicalsymptoms. In the last thirty years there is alack of information about AP incidence inRomanian apple orchards.To date, there are no studies about theoccurrence of AP in apple orchards fromRomania based on serological and/or moleculartests. To start to secure these missing data, apreliminary survey was carried out in 2012, intwo apple orchards located in Bistrita area,Romania, using serological and molecularassays.MATERIALS AND METHODS‘Florina’ cv., known to be one of the highlysusceptible to AP (Loi et al. 1995) and anothertwo Romanian cultivars, named ‘Generos’ and‘Aura’, were the subject of this study. Twentysamples of symptomatic apple trees werecollected in autumn 2012 from the threecultivars. Sampling was based on typical APsymptoms: witches’ brooms, foliar reddening inlate summer, enlarged stipules and dwarf offruits (Figure 1).Figure 1. Symptom of Apple proliferation disease: dwarfsized fruits (down) compared with healthy fruits (top) –original.Photo 1. Symptom of Apple proliferationdisease: dwarf sized fruits (down) comparedwith healthy fruits (top) – original.Serological diagnoses were performed byDouble Antibody Sandwich-Enzyme LinkedImmunosorbent Assay (DAS-ELISA) – (Clarkand Adams, 1977), using a monoclonalantibody (Loi et. al., 2002) raised against APphytoplasmas group, according to themanufacturer’s instructions (Bioreba,Switzerland). Absorbance values weremeasured at 405 nm after 30 and 60 minutes,using a TECAN plate reader. Samples wereconsidered positive if their absorbance valueswere more than twice those of negativecontrol. Positive and negative controls wereprovided in AP kit (Bioreba), and used both inserological and molecular assay.For molecular detection, total DNA waspurified by using DNeasy Plant Mini Kit andthe protocol recommended by manufacturer(Qiagen, Germany). DNA was extracted fromleaf veins and phloem, which were prior grindto a fine powder under liquid nitrogen. Aliquotsof DNA were then used in nested-PCR. A firstround of amplification was made by using anuniversal primers pair P1/P7 (Deng and Hiruki1991).RESULTS AND DISCUSSIONSSeventeen samples out of twenty reactedpositively by DAS-ELISA, using monoclonalantibody provided by Bioreba, whichspecifically recognize AP (Table 1).Nested-PCR, performed in parallel withserological detection, allowed us to detect16SrX phytoplasmas group. All samples testedby nested-PCR reacted positively, both in thefirst and the second PCR round.Consequently, “infected status” of all the 20trees showing typical AP symptoms analyzedin the present work was confirmed in nested-PCR.However, three samples were foundnegative in DAS-ELISA. There is possible thatthe three isolates were not recognized bymonoclonal antibody.Table 1. Results of serological and molecular testsCultivar Isolate code DAS-ELISA Nested-PCR StatusG1 + + infectedG2 + + infectedG3 + + infectedG4 + + infectedGenerosG5 + + infectedG6 + + infectedG7 + + infectedG8 + + infected144