Biological and Molecular Detection of Citrus Psorosis Virus in Citrus in the Eastern Mediterrenean Region of Turkey

Fifty bud sticks exhibiting vein flecking, vein clearing and oak-leaf pattern symptoms of ‘Satsuma’ and ‘Clementine’ mandarins, and ‘Washington navel’ and local oranges in the field in Mersin, Kozan and Adana provinces were collected and grafted on one-year-old sour orange seedlings to maintain Citrus psorosis virus (CPsV) in indicators. Thirty two indicator plants showed typical symptoms of vein flecking, vein clearing, and vein banding with oak-leaf pattern. Eighteen plants gave mild leaf symptoms. CPsV was detected by RT-PCR from CPsV-infected sources. Leaf samples for RT-PCR were collected from symptomatic field trees. In all, thirty samples from different trees of mandarin and twenty samples from different trees of sweet oranges were collected. A 434 by specific DNA for the coat protein was amplified from the cDNA of CPsV-infected leaf samples. This specific DNA product was not amplified from healthy leaf samples or asymptomatic leaves collected from CPsV-infected trees.     

Detection and characterization of citrus tatter leaf virus (CTLV) and citrus yellow vein clearing virus (CYVCV) in citrus trees from Cyprus

Citrus tatter leaf virus (CTLV) was firstly reported of in California. After that, it reported in Australia, Korea, Nigeria, Japan, South Africa, and China. The transmission of this virus from plant to plant is very easy with mechanically. Citrus yellow vein clearing virus (CYVCV) was reported on lemon trees in India, Pakistan, Turkey and China. Foliar distortion, necrotic spots, chlorosis and wrinkling symptoms were observed in young lemon orchards in newly established orchards with trees imported from abroad. Therefore, surveys of citrus trees in Cyprus were performed for CTLV and CYVCV from 2013 to 2016. A total of 64 leaf samples from symptomatic citrus trees (41 lemon, 10 orange, 10 mandarin and three grapefruit samples) were collected for total nucleic acid extraction and RT-PCR with CTLV primers to amplify a 309 bp and a 614 bp fragment, respectively, of the 5′ end (100%) and high nucleotide sequence identity (99%) with isolates BJNM-2 and QC4 from China and isolate BDZ-1 from Australia. To our knowledge, this is the first report of CTLV from Europe.     

Isolation of cytoplasts from Satsuma mandarin (Citrus unshiu Marc.) and production of alloplasmic hybrid calluses via cytoplast–protoplast fusion

Cytoplasm of Satsuma mandarin (Citrus unshiu Marc.) is known to influence seedlessness. Transfer of cytoplasm to a seedy cultivar could possibly lead to the production of seedless citrus fruits. In the present paper cytoplasts were isolated from cell suspension-derived protoplasts of Satsuma mandarin via ultra-centrifugation in a discontinuous gradient. No nucleus could be detected in the cytoplasts by DAPI (4′, 6-diamidino-2-phenylindole) staining compared with normal protoplasts. The cytoplasts, with high viability and small size, did not divide during solid embedding culture. Cytoplasts of Satsuma mandarin were electrically fused with embryogenic protoplasts of Murcott tangor (C. reticulata × C. sinensis), which led to regeneration of several cell lines. Flow cytometry (FCM) indicated that the cell lines were diploids. Simple sequence repeats (SSR) and cleaved amplified polymorphism sequence (CAPS) showed that the cell lines got their nuclear DNA from the protoplast parent, whereas the cytoplast parent donated the mtDNA, confirming transfer of mtDNA from Satsuma mandarin into Murcott tangor via cytoplast–protoplast fusion though no polymorphism was detected in chloroplast DNA between the fusion partners. This is the first report on isolation and characterization of cytoplasts, together with cytoplast–protoplast fusion in Citrus, which has a potential for citrus cultivar improvement involving cytoplasm transfer via cytoplast–protoplast fusion.     

Flower Formation from Citrus unshiu Buds Cultured In Vitro

Flowers were formed in vitro when buds of Satsuma mandarin (Citrusunshiu Mare) from the summer flush of growth harvested duringthe winter rest period before the onset of flower initiation,were grown on a solid Murashige and Skoog medium supplementedwith sucrose and a cytokinin. Flower development was dependentupon illumination, and was enhanced when a piece of stem wasattached to the bud. The percentage of flowering explants wasalways lower than the percentage of naturally flowering budsin spring, but treatments such as ringing which increase floweringin vitro, increased the number of explants flowering int vitroas well. Citrus unshiu Marc, ‘Satsuma’ mandarin, in vitro flowering, ringing     

The role as inoculum sources of Xanthomonas citri pv. citri surviving on the infected Satsuma mandarin fruits

Importing citrus fruits infected by Asiatic citrus canker caused by Xanthomonas citri pv. citri (Xcc) can act as an inoculum source for the disease epidemic in citrus canker-free countries. In this study, the pathogenicity of the causal agent of Asiatic citrus canker surviving on infected Satsuma mandarin fruits was evaluated. The washing solution of infected Satsuma mandarin fruits did not cause lesion formation on the citrus leaves. However, a typical citrus canker lesion was formed on the leaves after inoculation with higher concentrations of the inoculum from the washing solution (washing solution II). It indicated that the pathogenicity of the citrus canker surviving on the symptomatic Satsuma mandarin fruits was not changed. Scanning electron microscopic observation showed that the numbers of bacterial cells on the leaves of Satsuma mandarin which inoculated with the washing solution directly (washing solution I) was less compared to those of leaves inoculated with the washing solution II. This result spports that the pathogenicity of Xcc surviving on Satsuma mandarin fruits may not be changed but that the sucessful infection of citrus caker may depend on the concentration of the inoculum.     

Hormonal changes associated with fruit set and development in mandarins differing in their parthenocarpic ability

Satsuma [Citrus unshiu (Mak) Marc.] and Clementine [Citrus reticulata (Hort.) Ex. Tanaka, cv. Oroval] are two related species of seedless mandarins which differ in their tendency to set parthenocarpic fruits. Satsuma fruits naturally set parthenocarpically whereas Clementine mandarins show very low ability to set fruit in the absence of cross-pollination. The endogenous levels of gibberellins (GAs) and free and conjugated indole-acetic acid (IAA) and abscisic acid (ABA) throughout early stages of fruit development were investigated in seedless cultivars of both species. Analyses performed by full-scan combined gas chromatography-mass spectrometry (GC-MS) of extracts from ovaries at anthesis demonstrated the presence of GA₁₉, GA₂₀, GA₂₉, GA₁, GA₈, GA₃ and iso-GA₃ in Satsuma mandarin, whereas only GA₂₉, GA₃ and trace levels of GA₈ were detected in Clementine. At this developmental stage GA-like substances, as estimated by bioassay, reached their highest levels in Satsuma, while Clementine mandarins contained relatively lower levels. In both species the highest levels of free IAA were found at petal-fall stage at which time free ABA levels also peaked. Developing fruits of Clementine had higher amounts of both free IAA and ABA. In Satsuma, levels of conjugated IAA remained low throughout reproductive development whereas in Clementine they increased as the free form declined. In contrast, conjugated ABA was at low levels in Clementine but reached higher concentrations in Satsuma. These results suggest that in these mandarins the potential for setting parthenocarpic fruits is mainly influenced by the hormonal status of the fruit during the later stages of cell division and early stages of cell enlargement. Thus, the condition of low ability to set parthenocarpic fruits appears to be associated with lower levels of active GAs, lower capability to catabolize ABA to conjugated ABA and higher ability to conjugate IAA during this period.     

Low concentrations of NaHSO3 increase photosynthesis, biomass, and attenuate photoinhibition in Satsuma mandarin (Citrus unshiu Marc.) plants

Spraying low concentrated (0.5–5.0 mM) solutions of NaHSO₃ on Satsuma mandarin (Citrus unshiu Marc.) leaves resulted in enhancement (maximal about 15 % at 1 mM NaHSO₃) of net photosynthetic rate (P _N) for 6 d. The potential photochemical efficiency of photosystem 2 (PS2, F_v/F_m) and the quantum yield of PS2 electron transport (Φ_PS2) were increased under strong photon flux density (PFD). The slow phase of millisecond delayed light emission (ms-DLE) was increased, showing that the transmembrane proton motive force related to photophosphorylation was enhanced. We also observed that low concentrations of NaHSO₃ promoted the production of ATP in irradiated leaves. We suggest that the increase in P _N in Satsuma mandarin leaves caused by low concentrations of NaHSO₃ solution may have been due to the stimulation of photophosphorylation and, hence, the increase in photochemical efficiency through speeding-up of PS2 electron transport. Photoinhibition of photosynthesis in leaves was modified by NaHSO₃ treatment under high PFD. Hence the increase in leaf dry mass seems to be associated with the mitigation of photoinhibition caused by strong PFD.     

Production and molecular characterization of potential seedless cybrid plants between pollen sterile Satsuma mandarin and two seedy <Emphasis Type="Italic">Citrus</Emphasis> cultivars

Cytoplasmic male sterility (CMS) is known to be controlled by mitochondrial genome in higher plants including Satsuma mandarin (Citrus unshiu Marc.). Citrus symmetric fusion experiments often produce diploid cybrids possessing nuclear DNA from the mesophyll parent and mitochondrial DNA (mtDNA) from the embryogenic callus parent. Therefore, it is possible to transfer CMS from Satsuma mandarin as callus parent to seedy citrus cultivars as leaf one by somatic cybridization. Herein, symmetric fusion technique was adopted to create cybrids for potential seedlessness by transferring CMS from Citrus unshiu Marc. cv. Guoqing No. 1 (G1) to two traditional Chinese seedy citrus cultivars, ‘Shatian’ pummelo (C. grandis (L) Osbeck) and ‘Bingtang’ orange (C. sinensis (L) Osbeck). Flow cytometry analysis showed that 19 plants recovered from G1 + ‘Bingtang’ orange and 17 of 35 plants regenerated from G1 + ‘Shatian’ pummelo were diploid. The remaining plants from G1 + ‘Shatian’ pummelo were tetraploid. The diploid plants from the two combinations were confirmed as true cybrids by simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) analysis, with nuclear DNA from their corresponding leaf parent and mtDNA from their common suspension parent, G1 Satsuma mandarin. The remaining plants from G1 + ‘Shatian’ pummelo were identified as somatic hybrids with mtDNA from G1. The chloroplast simple sequence repeat (cp-SSR) analysis revealed somatic hybrid/cybrid plants from the two combinations in most cases possessed either of their parental chloroplast type, and two plants from G1 +‘Shatian’ pummelo and all embryoids analyzed from G1 + ‘Bingtang’ orange possessed chloroplast DNA (cpDNA) from both parents. These results demonstrated that we succeeded in introducing mtDNA from G1 Satsuma mandarin into the two target seedy citrus cultivars for potential seedlessness through symmetric fusion.     

The Influence of Ringing on Bud Development and Flowering in Satsuma Mandarin

Ringing of Satsuma mandarin (Citrus unshiu Marc.) trees showedincreased flowering in the following spring when performed duringSeptember and October, but not during November. Most of theeffect on flowering was due to an enhancement of both bud sproutingand the number of flowering shoots formed per node. In addition,a direct effect of ringing on flower initiation was demonstrated,since the number of vegetative shoots was reduced. The response of the buds to ringing was rapid as demonstratedby changes in bud weight, protein content, and electrophoreticpattern and behaviour when cultured in vitro. Buds from ringedtrees readily flowered in vitro when forced during the winterrest period and flower formation was enhanced by the additionof cytokinin. Buds from control trees formed a smaller numberof flowers in vitro, and flowering was much less enhanced bythe addition of cytokinin. It is concluded that ringing acceleratesflower initiation in the buds and this effect takes place beforethe winter rest period. Key words: Bud sprouting, Citrus unshiu Marc., flower initiation, flowering, in vitro flowering, Satsuma mandarin, ringing     

Abscisic acid increases in non-growing and paclobutrazol-treated fruits of seedless mandarins

Clementine ( Citrus reticulata [Hort.] Ex. Tanaka cv. Oroval) is a self-incompatible mandarin with a slow rate of fruit growth and high percentage of fruit abscission. Seedless Satsuma ( Citrus unshiu [Mak] Marc. cv. Clausellina) shows natural parthenocarpy and higher fruit set. Application of 25 μg fruit⁻¹ of paclobutrazol (PP333), an inhibitor of gibberellin biosynthesis, reduced the rate of growth and accelerated fruit abscission in both varieties. In contrast, gibberellin A₃ (GA₃) stimulated fruit growth only in the self-incompatible mandarin. Clementine fruits, in the absence of pollination, showed an approximately 2-fold transient increase in the free abscisic acid (ABA) content shortly after petal fall. In Satsuma, a very small accumulation of ABA was detected. Paclobutrazol treatment induced a 3-fold increase in ABA in Satsuma fruits but did not substantially affect the pattern of ABA accumulation in Clementine. In this variety, GA₃ suppressed the ABA increase observed in untreated fruits. These effects were observed 24 h after treatment. However, in Satsuma fruits, the effect of GA₃ on the ABA content was negligible. In addition, a comparative analysis of growing and non-growing fruits of Clementine showed that ABA, on a per unit weight basis, was always higher in the non-growing fruits. Treatment with 85 μM fluridone, an inhibitor of carotenoid biosynthesis and thus indirectly of ABA, delayed fruit abscission in Clementine, but also decreased fruit growth. Collectively, these observations indicate a relationship between high ABA content and a reduced rate of fruit growth and an acceleration of fruit abscission.     

Ethylene Production and Respiration of Postharvest Acid Citrus Fruits and Wase Satsuma Mandarin Fruit

Ethylene production and respiratory rate were examined in acid citrus fruits such as yuzu, seedless yuzu and daidai, and wase satsuma mandarin. A large amount of ethylene was produced from four varieties of citrus fruits harvested from May to July but not after September. A rise in ethylene production did not always coincide with a rise in respiratory rate. Excised tissues of fruits contained the ability of ethylene production throughout the developmental stages. The endogenous ethylene level at the ripening stage, reached the maximum when the color changed from green to yellow.     

Characterization of gamma-terpinene synthase from Citrus unshiu (Satsuma mandarin)

Gamma-terpinene is a monoterpene and a major component of essential oils made from citrus fruits and shows strong antioxidant activity in various assay systems. Plant gamma-terpinene synthase is a member of the monoterpene cyclase family, which produces a specific monoterpene through cyclization of geranyl diphosphate (GPP), but the monoterpene cyclases have not been fully characterized. It is necessary to prepare large amounts of gamma-terpinene synthase from Citrus unshiu (Satsuma mandarin) for the characterization, on this purpose we expressed the protein in Escherichia coli (E. coli) cells. As most monoterpene synthases have plastid-targeting signals, a gene lacking these signals was prepared and functionally expressed in E. coli cells harboring extra copies of the argU gene. The purified enzyme was incubated with GPP and the main product was confirmed to be gamma-terpinene by GC/MS.     

Flavonoids in Citrus and Related Genera

The occurence and distribution of flavonoid glycosides in young leaves and young and mature fruits of many citrus species and trifoliate orange were investigated. The occurence of neohesperidin in both young leaves and young fruits is fairly common to a number of species in subgenus Archicitrus. Ripe fruits of citrus could be classified into (a) the hesperidin group (b) the neohesperidin group (c) the naringin group and (d) the isonaringin group. A new flavanone glycoside, isonaringin, isolated from young fruits of Jagatarayu and Teng mikan is slightly bitter and has been determined by chemical and spectral evidences to have the structure of naringenin-7-rhamnoglucoside. Data showing the occurence of flavanone glycosides in some artificial citrus hybrids were also given.     

Integrative analyses of transcriptome and carotenoids profiling revealed molecular insight into variations in fruits color of Citrus Reticulata Blanco induced by transplantation

Citrus fruits exhibit vivid color and are favored extensively. However, the biochemical and molecular mechanism of Citrus Reticulata Blanco fruits coloring, especially the effect of transplantation on fruits coloring, is unclear. Herein, RNA-Seq and carotenoids profiling were applied to investigate the effect of transplantation on Orah mandarin fruits coloring. Transplantation induces fruit color shallowing, Ca²⁺ and ACC level declining and IAA level increasing. Transplantation induced variation in fruit skin and pulp carotenoids, mainly β-citraurin as one of the important pigments of citrus peel. 2253 up-regulated genes, 1103 down-regulated genes in skin and 815 up-regulated genes, 534 down-regulated genes in pulp of transplanted tree fruits were identified by RNA-Seq. The DEGs involved hormone signal, carotenoids biosynthesis and TFs such as MYB and bHLH family TFs. The carotenoid cleavage dioxygenase gene (Ciclev10028113m.g) is positively correlated with β-citraurin and regulated directly and/or indirectly by MYB1R1, PIF4, ACC and IAA. Integrative analyses revealed potential molecular insights into Orah mandarin peel color variation during transplantation.     

Symptoms induced by transgenic expression of p23 from Citrus tristeza virus in phloem‐associated cells of Mexican lime mimic virus infection without the aberrations accompanying constitutive expression

Citrus tristeza virus (CTV) is phloem restricted in natural citrus hosts. The 23‐kDa protein (p23) encoded by the virus is an RNA silencing suppressor and a pathogenicity determinant. The expression of p23, or its N‐terminal 157‐amino‐acid fragment comprising the zinc finger and flanking basic motifs, driven by the constitutive 35S promoter of cauliflower mosaic virus, induces CTV‐like symptoms and other aberrations in transgenic citrus. To better define the role of p23 in CTV pathogenesis, we compared the phenotypes of Mexican lime transformed with p23‐derived transgenes from the severe T36 and mild T317 CTV isolates under the control of the phloem‐specific promoter from Commelina yellow mottle virus (CoYMV) or the 35S promoter. Expression of the constructs restricted to the phloem induced a phenotype resembling CTV‐specific symptoms (vein clearing and necrosis, and stem pitting), but not the non‐specific aberrations (such as mature leaf epinasty and yellow pinpoints, growth cessation and apical necrosis) observed when p23 was ectopically expressed. Furthermore, vein necrosis and stem pitting in Mexican lime appeared to be specifically associated with p23 from T36. Phloem‐specific accumulation of the p23Δ158–209(T36) fragment was sufficient to induce the same anomalies, indicating that the region comprising the N‐terminal 157 amino acids of p23 is responsible (at least in part) for the vein clearing, stem pitting and, possibly, vein corking in this host.     

Effects of Exogenous Growth Regulators on Fruit Development in Citrus unshiu

The effects of applied growth regulators on fruit developmenthave been determined in the parthenocarpic Satsuma mandarin(Citrus unshiu Marc.). The application of either gibberellicacid or benzyladenine at flower opening, caused a transientincrease in cell division in the ovary wall, but had no significanteffect on final fruit size. Late fruit growth and final fruitsize were increased by the application of the synthetic auxin2,4,5-trichlorophenoxyacetic acid, which had a specific effecton the enlargement of the juice vesicles. The three growth regulators enhanced vascularization in thepedicel, but the growth effects observed were unrelated to theirinfluence on the transport capacity of the phloem but causedby their direct effects on the fruit tissues. The sensitivityof the fruit tissues to the applied growth regulators changedmarkedly during early fruitlet development, and was characterizedculturing the fruit tissues in vitro.Copyright 1993, 1999 AcademicPress Citrus unshiu Marc., fruit growth, hormone treatment, in vitro culture, phloem formation, phloem transport, xylogenesis     

Gibberellins and parthenocarpic ability in developing ovaries of seedless mandarins

Satsuma (Citrus unshiu [Mak] Marc.) and Clementine (Citrus reticulata [Hort.] Ex. Tanaka, cv Oroval) are two species of seedless mandarins differing in their tendency to develop parthenocarpic fruits. Satsuma is a male-sterile cultivar that shows a high degree of natural parthenocarpy and a high fruit set. Seedless Clementine varieties are self-incompatible, and in the absence of cross-pollination show a very low ability to set fruit. The gibberellins (GAs) GA53, putative 17-OH-GA₅₃, GA₄₄, GA₁₇, GA₁₉, GA₂₀, GA₂₉, GA₁, 3-epi-GA₁, GA₈, GA₂₄, GA₉, and GA₄ have been identified from developing fruits of both species by full-scan combined gas chromatography-mass spectrometry. Using selected ion monitoring with [²H₂]- and [¹³C]-labeled internal standards, the levels of GA₅₃, GA₄₄, GA₁₉, GA₂₀, GA₁, GA₈, GA₄, and GA₉ were determined in developing ovaries at anthesis and 7 days before and after anthesis, from both species. Except for GA8, levels of the 13-hydroxy-GAs were higher in Satsuma than in Clementine, and these differences were more prominent for developing young fruits. At petal fall, Satsuma had, on a nanograms per gram dry weight basis, higher levels of GA₅₃ (10.4x), GA₄₄ (13.9x), GA₁₉ (3.0x), GA₂₀ (11.2x), and GA₁ (2.0x). By contrast, levels of GA₈ were always higher in Clementine, whereas levels of GA₄ did not differ greatly. Levels of GA₉ were very low in both species. At petal fall, fruitlets of Satsuma and Clementine contained 65 and 13 picograms of GA₁, respectively. At this time, the application of 25 micrograms of paclobutrazol to fruits increased fruit abscission in both varieties. This effect was reversed by the simultaneous applications of 1 microgram of GA₃. GA₃ alone improved the set in Clementine (13x), but had little influence on Satsuma. Thus, seedless fruits of the self-incompatible Clementine mandarin may not have adequate GA levels for fruit set. Collectively, these results suggest that endogenous GA content in developing ovaries is the limiting factor controlling the parthenocarpic development of the fruits.