During several surveys in extensive areas in central Iran, apple trees showing phytoplasma diseases symptoms were observed. PCR tests using phytoplasma universal primer pairs P1A/P7A followed by ...R16F2n/R16R2 confirmed the association of phytoplasmas with symptomatic apple trees. Nested PCR using 16SrX group‐specific primer pair R16(X)F1/R1 and aster yellows group‐specific primer pairs rp(I)F1A/rp(I)R1A and fTufAy/rTufAy indicated that apple phytoplasmas in these regions did not belong to the apple proliferation group, whereas aster yellows group‐related phytoplasmas caused disease on some trees. Restriction fragment length polymorphism (RFLP) analyses using four restriction enzymes (HhaI, HpaII, HaeIII and RsaI) and sequence analyses of partial 16S rRNA and rp genes demonstrated that apple phytoplasma isolates in the centre of Iran are related to ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma aurantifolia’. This is the first report of apples infected with ‘Ca. Phytoplasma asteris’ in Iran and the first record from association of ‘Ca. Phytoplasma aurantifolia’ with apples worldwide.
In an effort to select and characterize apple rootstock resistant to apple proliferation (AP), progenies from seven apomictic rootstock selections and their parental apomictic species, Malus ...sieboldii and M. sargentii, were compared to standard stocks M 9 and M 11. Seedlings derived from open pollinated mother plants were grafted with cv. Golden Delicious and grown under natural infection conditions. The progenies differed greatly in resistance to the AP agent 'Candidatus Phytoplasma mali'. Progenies of M. sieboldii and its descendent rootstock selections D2212, 4608, 4551, and D1131 showed a high level of resistance, whereas progenies of M. sargentii and its descendent selections D1111 and C1828 proved susceptible. M 9 and M 11 showed an intermediate level of resistance. Phytoplasma titer in roots of the M. sieboldii and M. sargentii progeny groups was similarly low, whereas the concentration in the standard stocks was 100 to 5,000 times higher. In trees on most of the resistant stocks, only a minority was colonized in the scion, while in trees on susceptible and standard stocks, infection rate was often higher. Also, the titer in the top of trees on resistant stocks was usually lower than in trees on susceptible and standard stocks. Four progenies derived from open pollinated M. sieboldii and M. sieboldii descendents were subjected to DNA typing using simple sequence repeat (SSR) markers. This study revealed that the selected groups consisted mainly of mother-like plants (apomicts) and type I hybrids (unreduced mother genotype plus one male allele at each locus). Type II hybrids (full recombinants) and autopollinated offspring were rare. In the 4608 progeny, trees grown on type I hybrid rootstocks were significantly less affected than trees on mother-like stocks. In other progenies with fewer or no type I hybrids, trees on type II hybrids and autopollinated offspring suffered considerably more from disease than trees on mother-like stocks.
The effects of four commercially available bio-active compounds on the infection rates, symptom expression and growth rates of apple trees (Malus x domestica Borkh.) cv. Golden Delicious infected ...with 'Candidatus Phytoplasma mali' (the so-called Apple Proliferation phytoplasma or AP) were tested over a three-year period under controlled conditions. Post-infection treatments using Bion® (active ingredient: Acibenzolar-S-Methyl), Messenger® (Harpin protein), Regalis® (Prohexadione-Ca) and Dormex® (Cyanamide) had no significant effect on infection rates. Terminal growth of apple trees (grown as one-shoot pruned trees) was increased significantly by AP infection; Prohexadione-Ca was the only compound which had a significant (inhibiting) effect on the growth of both infected and non-infected apple trees. Acibenzolar-S-Methyl and Harpin had no significant effects on symptom expression. AP symptoms were masked during summer by Prohexadione-Ca, which caused severe growth abnormalities. Cyanamide changed the seasonal appearance of AP symptoms: while symptoms were delayed compared to the untreated control the first two years (2008 and 2009), symptoms appeared earlier the third year (2010). Differences in symptom expression levelled off later in the vegetative season, and no significant difference was found in October.
This study focused on evaluating the genetic diversity among ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) populations in orchards of north‐western Italy, where apple proliferation (AP) disease is ...widespread and induces severe economic losses. ‘Ca. P. mali’ was detected through restriction fragment length polymorphism (RFLP) analysis of PCR‐amplified 16S rDNA in 101 of 114 samples examined. Collective RFLP patterns, obtained by restriction analyses of four amplified genomic segments (16S/23S rDNA, PR‐1, PR‐2 and PR‐3 non‐ribosomal region, ribosomal protein genes rplV‐rpsC and secY gene), revealed the presence of 12 distinct genetic lineages among 60 selected representative ‘Ca. P. mali’ isolates, underscoring an unexpected high degree of genetic heterogeneity among AP phytoplasma populations in north‐western Italy. Prevalence of distinct genetic lineages in diverse geographic regions opens new interesting avenues for studying the epidemiology of AP disease. Furthermore, lineage‐specific molecular markers identified in this work could be useful for investigating the biological life cycle of ‘Ca. P. mali’.
Symptoms of pear decline (PD) were observed in several pear growing regions of Iran. Pear trees with typical symptoms of PD from Estahban (Fars Province) were examined for phytoplasma infection using ...polymerase chain reaction (PCR) assay. Graft inoculation of healthy pear trees with scions from diseased trees resulted in production of PD symptoms and transmission of phytoplasma as verified by PCR. Target DNA was amplified from symptomatic pear trees with fO1/rO1, an apple proliferation (AP) group-specific primer pair. Physical and putative restriction fragment length polymorphism (RFLP) analyses of fO1/rO1 primed PCR products showed profiles corresponding to AP group, 16SrX-C subgroup (Candidatus Phytoplasma pyri). Percent similarity values and phylogenetic analysis of fO1/rO1 primed sequences confirmed that, as a member of AP subclade, Estahban PD phytoplasma has a closer relationship to PD and peach yellow leaf roll phytoplasmas than to AP (Ca. Phytoplasma mali) and European stone fruit yellows (Ca. Phytoplasma prunorum) phytoplasmas. This is the first report of PD phytoplasma in the eastern Mediterranean.
The present paper describes a new approach for diagnosis of apple proliferation (AP) phytoplasma in plant material using a multiplex real-time PCR assay simultaneously amplifying a fragment of the ...pathogen 16S rRNA gene and the host,
Malus domestica, chloroplast gene coding for tRNA leucine. For the first time, such an approach, with an internal analytical control, is described in a diagnostic procedure for plant pathogenic phytoplasmas enabling distinction between uninfected plant material and false-negative results caused by PCR inhibition. Pathogen detection is based on the highly conserved 16S rRNA gene to ensure amplification of different AP phytoplasma strains. The newly designed primer/probe set allows specific detection of all examined AP strains, without amplifying other fruit tree phytoplasmas or more distantly related phytoplasma strains. Apart from its specificity, real-time PCR with serial dilutions of initial template DNA ranging over almost five orders of magnitude (undiluted to 80,000-fold diluted) demonstrated linear amplification over the whole range, while conventional PCR showed a reliable detection only up to 500-fold or 10,000-fold dilutions, respectively. Compared to existing analytical diagnostic procedures for phytoplasmas, a rapid, highly specific and highly sensitive diagnostic method becomes now available.
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