Characterization of a Phytoplasma Associated with Pear Decline in Iran

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.     

Dodder Transmission of Pear Decline, European Stone Fruit Yellows, Rubus Stunt, Picris echioides Yellows and Cotton Phyllody Phytoplasmas to Periwinkle

The pear decline, European stone fruit yellows and rubus stunt agents as well as the phytoplasmas causing Picris echioides (bristly oxtongue) yellows and cotton (Gossypium hirsutum) phyllody, respectively, were transmitted from naturally infected plants to the experimental host Catharanthus roseus (periwinkle) via dodder (Cuscuta spp.) bridges. The identities of the dodder-transmitted phytoplasmas were confirmed by restriction length fragment polymorphism analysis of polymerase chain reaction-amplified ribosomal DNA. On the basis of restriction profiles the cotton phyllody agent could be differentiated from the phytoplasma causing faba bean phyllody, a disease previously thought to be induced by the same organism as cotton phyllody.     

Isolation and characterization of polymorphic microsatellite markers from European pear (Pyrus communis L.)

This study reports the development and characterization of 19 microsatellite primer pairs developed from genomic DNA of European pear (Pyrus communis) and their transferability to other Pyrus and Malus material. The primers were designed from two different genomic libraries enriched for di‐ and trinucleotide repeats. When tested in six P. communis cultivars and 15 other Pyrus species, 13 primers revealed single‐locus polymorphism and six showed more complex patterns that suggest multiple loci. Two to 18 alleles were detected per locus and two primer pairs were sufficient to discriminate all accessions. Transferability of nine primer pairs to Malus was demonstrated through amplification of discrete products in two accessions.     

Effects of water stress on leaf growth and photosynthetic and transpiration rates of Tectona grandis

The response of pear fruit and leaf parameters to shade imposed during development was studied. Whole branches of mature trees of Pyrus communis L. cv. Bartlett growing in the High Valley area of Argentina were covered with a shade cloth (80 % reduction in irradiance) from 6 to 18 weeks after full bloom (WAFB) during the 1995-96 growing season. Fruit diameter was measured at two-weekly intervals; flesh firmness, soluble solids concentration, and leaf area were determined 18 WAFB. Prolonged shading significantly reduced fruiting spur specific leaf mass and consequently resulted in 20.79 % less final fruit fresh mass. However, flesh firmness was 8.07 % lower under full irradiance. This revised version was published online in July 2006 with corrections to the Cover Date.     

Changes in spectral properties of ageing and senescing maize and sunflower leaves

Activity and biochemical characteristic of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase from pear ( Pyrus communis cv. Blanquilla) was determined. The enzyme showed a low K_m (57.5 μM) for ACC and was dependent on O₂ (K_m 0.44% in atmosphere). It had an absolute requirement for Fe²⁺, ascorbate and CO₂ and was inhibited by α-aminoisobutyric acid (AIB: K₁ 4.2 m M ) and cobalt. ACC oxidase has an optimum pH of 6.7 and temperature maxima at 28 and 38°C and it is concluded that the activity of ACC oxidase from pear resembles authentic in vivo activity.     

香梨果实成熟衰老过程中4种内源激素的变化

以库尔勒香梨[白梨(PyrusbretschneideriRehd.)的变种]为材料,在果实生长发育、成熟衰老期间检测内源IAA、GA3、ABA、乙烯含量变化规律及其相互关系。结果表明果实发育初期IAA、GA3、ABA含量最高,有利于幼果坐果;CA3与ABA的比值变化对果实迅速膨大起关键作用;高浓度GA3对阻抑叶绿素分解起明显作用;果实成熟衰老期间,IAA含量与乙烯释放速率呈方向相同的变化;在此期间GA3含量变化与乙烯释放变化相反。     

Ripening-related changes in the cell walls of Spanish pear (Pyrus communis)

Unripe Spanish pears ( Pyras commanis L. ev. Blanquilla ) were ripened at 18°C for 5 and 10 days. Softening of the cortical tissues was associated with swelling of parenchyma cell walls from 1 to more than 5 μm in 10 day ripe pears, by which time the pears were over ripe. However, there was little indication of cell separation and the middle lamella could be detected between most cell walls. Furthermore, cell separation was constrained by regions rich in plasmodesmata where wall swelling was prevented. Parenchyma cells in the 500 μm of tissue underlying the epidermis did not undergo ripening-related changes to the same extent as those of the cortex. These cells, in combination with a sub-epidermal layer of lignified sclereid clusters, constituted a relatively tough and protective skin. Ripening of the cortical tissues was associated with a depletion of alcohol-insoluble pectic polysaccharides, as indicated by the decrease in arabinose and uronic acid. Analysis of alcohol-insoluble cell wall preparations enriched in either parenchyma or sclereid cell walls indicated that this change was predominantly associated with the parenchyma walls. Such changes were less prominent in the peel. The decrease in pectic polysaccharides was accompanied by an increase in their solubility. During ripening, the sclereid clusters of the cortex continued in develop, as indicated by an increase in their size and yield of cell wall xylose and glucose. Cortical parenchyma cells radiating from the sclereids were firmly attached to the lignified cells. This was due to lignification extending from the sclereids into the primary walls of the parenchyma cells. We conclude that dissolution of pectic polysaccharides is one of several factors which determine softening during ripening of Spanish pears.     

PCR detection of MLOs in quick decline-affected pear trees in Italy

Polymerase chain reaction (PCR) amplification, using primers derived from the 16S rRNA gene, followed by restriction fragment length polymorphism (RFLP) analysis with Alu I restriction endonuclease was used to detect myc-oplasma-like organisms (MLOs) associated with pear decline. MLOs were consistently detected in pear trees that suddenly wilted and died within a few days during summer, as well as in pears of the same orchards with symptoms similar to the slow form of pear decline. In both cases the same RFLP pattern was obtained. Declining pear trees were 5 to 8-yr-old cvs Williams, Kaiser and Max Red Bartlett grafted on to Pyrus communis seedling rootstocks. All the orchards affected by quick decline had severe attacks of pear psyllid (Cacopsylla pyri) during the year this study was performed and during the previous year. The results showed the suitability of DNA amplification by the polymerase chain reaction for the detection of pear decline MLOs and established that MLOs can be detected in infected tissues of dead trees.     

梨多倍体化对离体叶片不定梢再生能力的影响

以源于二倍体梨品种Fertility(Pyrus communis L.)通过秋水仙碱离体诱变体细胞染色体加倍获得的不同同源多倍体无性系为试材,以离体叶片为外植体,观察研究了不同倍性无性系叶片的不定梢再生能力。结果表明,多倍体的不定梢再生率显著低于二倍体的再生率。不同多倍体无性系的不定梢再生能力也存在显著差异。三倍体无性系3x-3和四倍体无性系4x-4不能诱导产生不定梢。表明器官发生能力下降或植物细胞全能性的丧失与细胞染色体多倍体化有关。     

Changes in tonoplast protein and density with the development of pear fruit

Protoplasts isolated from pear fruit at the end of the cell‐division stage, 30 days after flowering (DAF), had already formed a large central vacuole and the vacuole occupied most of the protoplast. The changes in protein composition and density of the tonoplast (vacuolar membrane) were investigated during fruit development. After a linear sucrose density gradient centrifugation, the distribution of tonoplasts at 30 and 48 DAF was broad and began to narrow with further fruit development. This suggests that the tonoplast of young fruit is heterogeneous and becomes homogeneous with fruit development. The apparent density of the tonoplast at 30 DAF was approximately 1.12 g ml⁻¹; it decreased with fruit development and was finally 1.09 g ml⁻¹ in mature fruit. The phospholipid amount on the basis of tonoplast protein was 0.80 mg mg⁻¹ at 30 DAF. It increased with fruit development, and finally reached 7.49 mg mg⁻¹. This result indicates that the decrease in the density of the tonoplast was caused by the increase in the ratio of phospholipid to membrane protein. The protein composition of the tonoplast at each stage was quite different. The level of polypeptides of 94, 70, 61, 52, 48 and 41 kDa was low in young fruit and high in the middle or later stages of fruit development. In contrast, the level of a 76‐kDa polypeptide was high in young fruit and decreased with fruit development. Although their functions are still unclear, these tonoplast proteins may play important roles in fruit development.     

Iron deficiency-induced changes in the photosynthetic pigment composition of field-grown pear (Pyrus communis L) leaves

In this work we characterize the changes induced by iron deficiency in the pigment composition of pear (Pyrus communis L.) leaves grown under high light intensities in field conditions in Spain. Iron deficiency induced decreases in neoxanthin and β-carotene concomitantly with decreases in chlorophyll a, whereas lutein and carotenoids within the xanthophyll cycle were less affected. Iron deficiency caused major increases in the lutein/chlorophyll a and xanthophyll cycle pigments/chlorophyll a molar ratios. The chlorophyll a/chlorophyll b ratio increased in response to iron deficiency. The carotenoids within the xanthophyll cycle in iron-deficient and in iron-sufficient (control) leaves underwent epoxidations and de-epoxidations in response to ambient light conditions. In control leaves dark-adapted for several hours, most of the xanthophyll cycle pigment pool was in the epoxidated form vio-laxanthin, whereas iron-deficient leaves had significant amounts of zeaxanthin. Iron-deficient leaves also exhibited an increased non-photochemical quenching, supporting the possibility of a role for pigments within the xanthophyll cycle in photoprotection.     

Change in chemical constituents and free radical-scavenging activity during Pear (Pyrus pyrifolia) cultivar fruit development

Changes in chemical constituent contents and DPPH radical-scavenging activity in fruits of pear (Pyrus pyrifolia) cultivars during the development were investigated. The fruits of seven cultivars (cv. Niitaka, Chuhwangbae, Wonhwang, Hwangkeumbae, Hwasan, Manpungbae, and Imamuraaki) were collected at 15-day intervals after day 20 of florescence. Vitamins (ascorbic acid and α-tocopherol), arbutin, chlorogenic acid, malaxinic acid, total caffeic acid, total flavonoids, and total phenolics were the highest in immature pear fruit on day 20 after florescence among samples at different growth stages. All of these compounds decreased gradually in the fruit during the development. Immature pear fruit on day 35 or 50 after florescence exhibited higher free radical-scavenging activity than that at other times, although activities were slightly different among cultivars. The chemical constituent contents and free radical-scavenging activity were largely different among immature fruits of the pear cultivars, but small differences were observed when they matured.     

Influence of UV-B radiation on developmental changes, ethylene, CO2 flux and polyamines in cv. Doyenne d'Hiver pear shoots grown in vitro

In vitro shoots of cv. Doyenne ďHiver pear ( Pyrus communis L.) were irradiated under controlled environments for 6 h per day at 5 different levels of biologically effective UV-B radiation (UV-B_BE). UV-B exposure caused a progressive increase in apical necrosis above background levels and stimulated leaf abscission. Shoots grown for 2 weeks at 7. 8 mol m⁻² day ⁻¹ of photosynthetic photon flux (PPF) and treated with 8. 4 or 12. 0 kJ m⁻² day ⁻¹ UV-B_BE produced up to 4 times more ethylene than those given 2. 2 or 5. 1 kJ m⁻² day⁻¹ UV-B_BE or untreated controls. Exposure of shoots to 12 kJ m⁻² day ⁻¹ of UV-B_BE caused an increase in free putreseine content after 4 to 14 days of irradiation. Shoots showed a decrease in CO₂ uptake after 3 days of UV-B: thereafter, they appeared to recover their photosynthetic capacity. Under typical PPF conditions used in micropropagation (90 μmol m⁻² S⁻¹). 8. 4 kJ m⁻² day ⁻¹ of UV-B radiation was injurious to realatively tender tissues of in vitro pear shoots: increasing the level of UV-B_BE to 12 kJ m⁻² day⁻¹ produced even more adverse effects.     

Molecular Characterization of Phytoplasmas Related to Apple Proliferation and Aster Yellows Groups Associated with Pear Decline Disease in Iran

Pear trees showing pear decline disease symptoms were observed in pear orchards in the centre and north of Iran. Detection of phytoplasmas using universal primer pair P1A/P7A followed by primer pair R16F2n/R16R2 in nested PCR confirmed association of phytoplasmas with diseased pear trees. However, PCR using group‐specific primer pairs R16(X)F1/R16(X)R1 and rp(I)F1A/rp(I)R1A showed that Iranian pear phytoplasmas are related to apple proliferation and aster yellows groups. Moreover, PCR results using primer pair ESFYf/ESFYr specific to 16SrX‐B subgroup indicated that ‘Ca. Phytoplasma prunorum’ is associated with pear decline disease in the north of Iran. RFLP analyses using HaeIII, HhaI, HinfI, HpaII and RsaI restriction enzymes confirmed the PCR results. Partial 16S rRNA, imp, rp and secY genes sequence analyses approved that ‘Ca. Phytoplasma pyri’ and ‘Ca. Phytoplasma asteris’ cause pear decline disease in the centre of Iran, whereas ‘Ca. Phytoplasma prunorum’ causes disease in the north of Iran. This is the first report of the association of ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma prunorum’ with pear decline disease worldwide.     

Isolation and characterization of microsatellite markers in Japanese pear (Pyrus pyrifolia Nakai)

Fourteen microsatellite markers were developed from an enriched genomic library of Japanese pear (Pyrus pyrifolia) by selective hybridization. They were characterized using 17 Japanese pear cultivars. The expected heterozygosity and observed heterozygosity ranged from 0.21 to 0.74 and from 0 to 0.88, respectively. Two to 11 alleles were detected per locus, with IPPN09 and IPPN15 judged to amplify multiple loci. IPPN17 was the most informative locus with the lowest probability of identity (0.19). These primers exhibited a high cross‐species transferability between species and genera.     

梨实生树不同发育区的叶片细胞学研究

采用显微分光光度计和显微图像分析仪比较研究了8年生梨实生树(Pyrus pyrifliaNakai)童区和成年区叶片细胞核 DNA含量、RNA含量和细胞、细胞核面积大小的差异。梨实生树从童区向成年区转变后,叶片内细胞核DNA含量上升,细胞内RNA合成加强,细胞和细胞核面积增大;同时,叶肉组织结构分化程度提高,叶面积增大,叶片加厚。     

西洋梨水孔蛋白基因家族的全基因组鉴定及表达分析

水孔蛋白(AQPs,aquaporins)是高效转运水分子的膜内在蛋白,具有丰富的多样性,在调控植物的水分关系中有重要作用。本研究利用西洋梨(Pyrus communis L.‘Bartlett’)基因组数据库,通过生物信息学手段鉴定西洋梨PcAQPs基因家族成员;并利用MEGA 6.0.5软件,采用邻接法构建系统发育树;利用GSDS 2.0软件进行基因结构分析,MEME程序进行Motif分析,AgBase v2.00程序进行GO分析;采用半定量RT-PCR技术研究PcAQPs基因组织表达情况。结果表明,西洋梨基因组中共有54个PcAQPs家族成员,均含有AQP特征结构域和保守的Motif基序,根据基因结构及系统进化分析可分为PIP、TIP、NIP和SIP等4个亚家族。不同基因间结构差别较大,但聚类关系较近的基因其结构类似。GO分析发现,多数PcAQPs基因具有转运蛋白活性,参与物质转运、应激反应、发育和代谢等生物学过程,但不同亚家族成员构成的细胞组分及参与的生物学过程具有明显差异。半定量结果表明,大多数PcAQPs基因在根、茎、叶和果实中均有表达,而且不同基因家族、不同基因间的组织表达模式存在差异。该研究为今后西洋梨PcAQPs基因的克隆和功能分析奠定了基础。     

Response of the pear rootstock to boron and salinity in vitro

The effects of boron and NaCl induced salinity on growth and mineral composition of the pear (Pyrus communis L.) rootstock OH × F 333 shoots cultured in vitro were investigated. Shoots were grown in vitro for seven weeks on a Murashige and Skoog medium containing two B concentrations (0.1 and 2 mM) combined with five NaCl concentrations (0, 10, 20, 40, and 80 mM). The longest shoots were produced at 0.1 mM B and 80 mM NaCl, but highest number of shoots were produced at 0.1 mM B and 0–20 mM NaCl. Inclusion of 20 and 40 mM NaCl in the culture medium significantly increased fresh mass of cultures compared to 0 mM NaCl for all B concentrations tested. The concentrations of P, K, Ca, Mg, Na, Fe, Mn and Zn of plants were affected by B and NaCl concentration of the medium.     

Detection and quantification of four species of the genus Clostridium in infant feces

To determine the composition of Clostridium in the feces of infants approximately 30 days old, we have developed a detection and quantification method of Clostridium paraputrificum, Clostridium perfringens, Clostridium tertium, and Clostridium difficile by species-specific primers. C. perfringens and C. difficile were detected in four fecal samples from 22 infants (18.2%), whereas C. paraputrificum was detected in three samples (16.7%). C. tertium was detected in two samples (9.1%). Moreover, the occurrences of the four species in bottle-and mix-fed infants were relatively higher than in breast-fed infants (P< 0.05). Subsequently, positive samples detected by nested PCR (polymerase chain reaction) were subjected to realtime PCR. The results showed that the numbers of C. paraputrificum, C. perfringens, C. tertium, and C. difficile ranged from about 1x10(5) to 3x10(7) cells/g wet feces.