Localisation and movement of Plum pox virus in apricot stem tissues

Localisation and movement of Plum pox virus (PPV), sharka disease, in stem tissues of susceptible and resistant apricot (Prunus armeniaca L.) cultivars was studied. Two different assays were performed. In the first assay, apricot cultivars were grafted on to a non‐inoculated GF305 peach rootstock and, after two months, the sprouted apricot was inoculated by chip‐grafting. In the second assay, apricot cultivars were grafted on to a previously chip‐inoculated GF305 showing strong PPV symptoms. Localisation of virus was studied in apricot stem by immuno‐tissue printing and sharka symptoms in GF305 and apricot leaves were also observed. Virus was mainly localised in the xylem, and sometimes in the cortex and pith. Results revealed that, while all the cultivars allowed limited virus movement from the inoculation point, only the susceptible cultivars (Screara, Bebeco and Colomer) allowed long distance movement and even showed symptoms in leaves.     

Flanking the major Plum pox virus resistance locus in apricot with co-dominant markers (SSRs) derived from candidate resistance genes

Plum pox virus (PPV) is a potyvirus that causes sharka disease in infested stone fruit trees (Prunus species, peach, apricot, plum). In apricots, the resistance is controlled by a major quantitative trait locus that explains up to 70% of the phenotypic variance; it is localised in the upper part of linkage group 1. In this report, we transformed candidate genes that mapped in the region of the apricot resistance locus into polymerase chain reaction markers (SSCP and SSR) and tested for their co-localisation with the major PPV resistance locus in related and unrelated populations. Three populations of F1 and F2 individuals issued from crosses between the PPV-resistant cultivar ‘Stark Early Orange’ or ‘Goldrich’ and three susceptible parents were used in this study. Molecular-marker data were collected to determine the linkage relationship between the PPV resistance locus in apricots and markers that target candidate disease-resistance genes. In addition, SSR markers linked to resistance-gene candidates were mapped to positions flanking the PPV resistance locus in different apricot populations. Therefore, we demonstrate that this strategy helps to saturate the major genomic region controlling resistance to PPV in apricot with valuable co-dominant markers. O. Sicard and G. Marandel have contributed equally to this work.     

Inheritance of resistance to Plum pox virus in apricot (Prunus armeniaca L.)

The inheritance of resistance to Plum pox virus (PPV) has been studied in 1,178 apricot hybrids. Seven hundred and eighteen F1 hybrids, obtained from controlled crosses between the susceptible Greek cultivar “Bebecou” and the resistant PPV cultivars of American origin (“Stark Early Orange,” ‘NJA2,” ‘Veecot,” “Sunglo,” “Harlayne,” and “Orangered”) were evaluated for resistance to the PPV-M (Marcus) strain, 8 years after artificial inoculation. The inheritance of resistance to PPV has been additionally studied for the first time in a BC₁ population of 95 apricot hybrids for four vegetative periods. Reaction of each hybrid to PPV-M was scored through visual symptoms, indexing onto GF-305 and double-antibody sandwich enzyme-linked immunosorbent assay tests. Segregation within the hybrids, determined by Chi-squared analysis, fits a 1:1 ratio (P ≤ 0.05) of the resistant vs susceptible, indicating that resistance to PPV is controlled by a single dominant gene locus and that the above six resistant cultivars are heterozygous for the trait. Plants carrying this gene may initially develop disease symptoms on leaves but eventually recover and no virus can be detected in leaves. Susceptible plants show similar symptoms initially but remain symptomatic. Inheritance of resistance to PPV also has been studied in 365 F1 hybrids by crossing the resistant cultivar “Stella” with the susceptible “Bebecou” and the resistant cultivars “Sunglo” and “NJA2,” for 8 years after inoculation. The segregation ratio was 1:0 (resistant/susceptible) suggesting that “Stella” is homozygous for the resistance trait. The purpose of this work was the enhancement of the knowledge of inheritance of resistance to PPV for the selection of new cultivars.     

Interspecific transfer of resistance to Plum pox virus from almond to peach by grafting

Grafting almond variety ‘Garrigues’ onto ‘GF305’ peach seedlings heavily infected by Plum pox virus (PPV) progressively produces the disappearance of viral symptoms and drastically reduces virus accumulation in ‘GF305’ rootstock, in most cases to undetectable levels. This response appears to be specific between almond and peach, as it was not consistently observed by grafting ‘Garrigues’ onto other Prunus species such as plum (‘Adesoto’) or apricot (‘Real Fino’). The ability to induce resistance to PPV in ‘GF305’ was transmitted to the sexual descendants of Garrigues. Furthermore, grafting ‘Garrigues’ onto ‘GF305’ before PPV inoculation completely prevented virus infection, showing that the resistance is constitutive and not induced by the virus. This fact suggests that resistance may be due to the transfer of a defence factor from ‘Garrigues’ almond through the graft union and its interaction with specific factors of ‘GF305’ peach to produce the antiviral response. These results open new avenues to potential protection against PPV in peach, the most economically important species among stone fruits.     

Intra-strain biological and epidemiological characterization of plum pox virus

Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection.     

Generation and characterisation of functional recombinant antibody fragments against RNA replicase NIb from plum pox virus

A monoclonal antibody (mAb 2A) able to react against the RNA replicase NIb from plum pox virus (PPV) was obtained and used for generating a specific scFv fragment. The VH and VL coding sequences were cloned and expressed as a fusion scFv protein to alkaline phosphatase. This fusion protein was able to recognise viral NIb in both Western and tissue-print ELISA blots. The affinity and specificity of scFv2A for NIb was similar to that of the parental mAb and the region YLEAFY from PPV-NIb was identified by PEPSCAN assay as the putative epitope. Isolated VH domains from scFv2A were also expressed as fusion to alkaline phosphatase. However, their ability to react against NIb was greatly altered. scFv2A fragments were transiently expressed in the cytosol of Nicotiana benthamiana and although they accumulated to low levels, inhibition-ELISA results indicated that they retained antigen-binding activity.     

Identification of biomechanical factors involved in stem shape variability between apricot tree varieties

BACKGROUND AND AIMS: Stem shape in angiosperms depends on several growth traits such as elongation direction, amount and position of axillary loads, stem dimensions, wood elasticity, radial growth dynamics and active re-orientation due to tension wood. This paper analyses the relationship between these biomechanical factors and stem shape variability. METHODS: Three apricot tree varieties with contrasting stem shape were studied. Growth and bending dynamics, mechanical properties and amount of tension wood were measured on 40 1-year-old stems of each variety during one growth season. Formulae derived from simple biomechanical models are proposed to quantify the relationship between biomechanical factors and re-orientation of the stems. The effect of biomechanical factors is quantified combining their mechanical sensitivity and their actual variability. RESULTS: Re-orientations happened in three main periods, involving distinct biomechanical phenomena: (a) passive bending due to the increase of shoot and fruit load at the start of the season; (b) passive uprighting at the fall of fruits; (c) active uprighting due tension wood production at the end of the season. Differences between varieties mainly happened during periods (a) and (b). CONCLUSIONS: The main factors causing differences between varieties are the length/diameter and the load/cross-sectional area ratios during period (a). Wood elasticity does not play an important role because of its low inter-variety variability. Differences during period (b) are related to the dynamics of radial growth: varieties with early radial growth bend weakly upward because the new wood layers tend to set them in a bent position. The action of tension wood during period (c) is low when compared with passive phenomena involved in periods (a) and (b).     

The influence of polyamines on apricot ovary development and fruit set

Polyamine (PA) concentrations of ovaries of two apricot cultivars, ‘S405‐17’ and ‘Bergeron’, collected at three stages of floral development, were analysed. The relationships between PA profiles, ovule development and fruit load between the two apricot cultivars were also determined. We show that PA levels vary with ovary development, depending on the cultivar. Higher concentrations of putrescine (PUT), spermidine and spermine were found in S405‐17 cultivar at all phenological stages. In addition, S405‐17 cultivar had more developed ovules and a higher fruit load than Bergeron. Moreover, the exogenous application of 10 mM PUT in Bergeron increased the percentage of functional ovules from 17.1% (control) to 33.3%. These results prove the effects of PAs on ovary development and indicate the possible improvement of apricot ovule development by their exogenous application. All these results define the relationship between PA content and ovule development in apricot. Further studies would confirm the use of free endogenous PA concentrations as markers of the developmental stage of the ovary, because determination of PA contents is easier and faster than serial sectioning.     

Benzothiadiazole and l-2-oxothiazolidine-4-carboxylic acid reduce the severity of Sharka symptoms in pea leaves: effect on antioxidative metabolism at the subcellular level

The effect of treatment with benzothiadiazole (BTH) or l -2-oxothiazolidine-4-carboxylic acid (OTC), and their interaction with Plum pox virus (PPV) infection, on antioxidative metabolism of pea plants was studied at the subcellular level. PPV infection produced a 20% reduction in plant growth. Pre-treatment of pea plants with OTC or BTH afforded partial protection against PPV infection, measured as the percentage of leaves showing symptoms, but neither BTH nor OTC significantly reduced the virus content. PPV infection caused oxidative stress, as monitored by increases in lipid peroxidation and protein oxidation in soluble and chloroplastic fractions. In leaves of non-infected plants, OTC increased the content of reduced glutathione (GSH) and total glutathione; accordingly, an increase in the redox state of glutathione was observed. An increase in oxidized glutathione (GSSG) was found in symptomatic leaves from infected plants. A similar increase in GSSG was also observed in asymptomatic leaves from infected, untreated plants. However, no changes in GSSG occurred in asymptomatic leaves from infected plants treated with BTH and OTC and, accordingly, a higher redox state of GSH was recorded in those leaves, which could have had a role in the reduction of symptoms, as observed in asymptomatic leaves from infected plants treated with BTH or OTC. Treatment with BTH or OTC had some effect on antioxidant enzymes in soluble and chloroplastic fractions from infected pea leaves. An increase in antioxidative mechanisms, such as GSH-related enzymes (DHAR, GR and G6PDH), as well as APX and POX, at the subcellular level was observed, which could play a role in reducing the severity of cellular damage induced by Sharka in pea leaves.     

Plum Pox Virus in Japanese Plum from Argentina: Serological Detection and Molecular Characterization of an Isolate from cv. Red Beauty

A Plum pox virus (PPV) isolate detected in a Japanese plum orchard in Pocito (San Juan, Argentina) was transmitted mechanically to Prunus tomentosa and Nicotiana benthamiana. DAS‐ELISA and DASI‐ELISA indicated the virus presence and serological relationship with D‐strain isolates; IC‐RT‐PCR amplified a 1.2‐kb fragment of the virus genome encoding the CP‐3′ nc region. The analysis of the sequence showed the presence of the DAG motif at the 5′ end of the capsid protein and the Rsa I and Alu I sites at the 3′ end. The phylogenetic relationships and multiple alignment with PPV isolates from NCBI database indicated greatest (+98%) homology with the D strain and close identity with MNAT1 ( AF360579 ) USA peach isolate. The sequence analysed showed two amino acid mutations towards the 5′ N‐terminus of CP (the most variable region) with respect to a consensus of PPV D‐strain isolates. This is the first molecular characterization of 3′terminal genome region of PPV isolate to confirm D strain in a Japanese plum from Argentina.     

Improved efficiency in apricot breeding: Effects of embryo development and nutrient media on in vitro germination and seedling establishment

Apricot (Prunus armeniaca L.) embryos at three stages of development were cultured on C2d, SBH and WPM media. In vitro culture produced high percentages of germination and seedlings throughout all three developmental stages. Significant media effects were noted for changes in both embryo length and weight during the culture period, as well as number of plants produced. Embryos between 5 and 9 mm (developmental stage I) germinated and developed into plants in a significantly higher percentage than in the other two more mature stages. Therefore, embryo culture can be successfully used as a tool in an apricot breeding program to obtain higher percentages of seedlings from planned hybridization or to overcome a lack of seed germination.     

Typing Primus necrotic ringspot virus isolates by serology and restriction endonuclease analysis of PCR products

Primus necrotic ringspot virus (PNRSV) isolates were characterised by bioassays, serotyping and restriction fragment length polymorphism (RFLP) analysis of PCR products. Based on symptoms in host trees and bioassays it was concluded that only one of the 16 tested isolates is severe. The serotyping results demonstrated that by using four different MAbs in TAS-ELISA the tested isolates could be divided into four subgroups, however, the severe isolate could not be singled out. RFLP analysis of PCR products supported the serotyping data but did not differentiate between isolates of the two main serological subgroups. A restriction map, derived from sequence analysis of the PCR products obtained from selected isolates, allowed exact location of the restriction sites within the PCR products of each isolate. A mild isolate with a unique genome structure was identified by both serological and RFLP assays. As far as we are aware, this is the first report on sub-grouping of PNRSV isolates by bioassays, serotyping with MAbs and RFLP analysis.     

Preparation of novel authentication film by screen printing of anthocyanin biomolecular extract: Thermochromism and vapochromism

Novel thermochromic and vapochromic paper substrates were prepared via screen printing with anthocyanin extract in the presence of ferrous sulfate mordant, resulting in multi-stimuli responsive colorimetric paper sheets. Environmentally friendly anthocyanin extract was obtained from red-cabbage (Brassica oleracea var. capitata L.) to function as spectroscopic probe in coordination with ferrous sulfate mordant. Pink anthocyanin/resin nanocomposite films immobilized onto paper surface were developed by well-dispersion of anthocyanin extract as a colorimetric probe in a binding agent without agglomeration. As demonstrated by CIE colorimetric studies, the pink (λ_max = 418 nm) film deposited onto paper surface turns greenish-yellow (λ_max = 552 nm) upon heating from 25 to 75°C, demonstrating new thermochromic film for anti-counterfeiting applications. The thermochromic effects were investigated at different concentrations of the anthocyanin extract. Upon exposure to ammonia gas, the color of the anthocyanin-printed sheets changes rapidly from pink to greenish-yellow, and then immediately returns to pink after taking the gaseous ammonia stimulus away, demonstrating vapochromic effect. The current sensor strip showed a detection limit for ammonia gas in the range 50–300 ppm. Both thermochromism and vapochromism showed high reversibility without fatigue. In addition to studying the rheological properties of the prepared composites, the morphological and mechanical properties of the printed cellulose substrates were also studied.     

Restoration of vigor and rooting competence in stem tissues of mature citrus by repeated grafting of their shoot apices onto freshly germinated seedlings in vitro

Summary Repeated grafting of 0.2-cm shoot tips from fruiting-age trees ofCitrus reticulata Blanco ‘Ponkan’ mandarin andC. sinensis Osbeck ‘Liu Tseng’ sweet orange onto freshly germinated ‘Troyer’ citrange [Poncirus trifoliata (L.) Raf. X.C. sinensis Osbeck] seedlings in vitro resulted in progressive restoration of rooting competence and vigor of regenerated roots and shoots. The restored traits were retained through the course of the investigation and suggested a phase reversal phenomenon.     

Effects of ribavirin and adenine arabinoside on tobacco mosaic virus in Nicotiana tabacum L. var Xanthi tissue cultures

Although both ribavirin (1--ribofuranosyl-1,2,4-triazole-3carboxamide) and adenine arabinoside inhibited the multiplication of tobacco mosaic virus (TMV) in mechanically inoculated leaf tissues, neither chemical inhibited virus multiplication in unorganized tobacco callus after in vitro inoculation. The adenine deaminase inhibitor, pentostatin, did not increase the activity of adenine arabinoside in cultured cells. Several different developmental conditions and media did not increase the ability of either chemical to eradicate the virus from tobacco tissue cultures. However, the virus was eradicated from TMV-infected callus when grown in the presence of combinations of ribavirin and adenine arabinoside in shoot inducing medium.