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PubMed2000

Specific IgG1 and IgG4 antibodies to citrus red mite in citrus farmers: a study of their relationship to respiratory symptoms.

Citrus red mite (CRM) is known as the most common sensitizing allergen in subjects with asthma and rhinitis working on citrus farms. The aim of this study is to evaluate the role of specific IgG1 (slgG1) and specific IgG4 (slgG4) to CRM in citrus farmers. Questionnaire survey and skin prick test including CRM antigen was done by 136 workers. Specific IgE (slgE), slgG1 and slgG4 to CRM were detected by enzyme-linked immunosorbent assay (ELISA). CRM-sensitive-asthma was diagnosed upon presence of asthmatic symptoms by questionnaire, airway hyperresponsiveness to methacholine and slgE to CRM. CRM-sensitive rhinitis was diagnosed upon presence of rhinitis symptoms and slgE to CRM. Eleven (8.1%) had CRM-sensitive asthma and 25 (18.4%) had CRM-sensitive rhinitis. Significant association was noted between presence of asthmatic symptoms and slgE or slgG4 (p<0.05, respectively), while no significant association was noted in slgG1 (p>0.05). Significant association was noted in the prevalence between slgG4 and slgE (p<0.05), while no significant association was noted between slgG1 and slgG4 or slgE (p<0.05, respectively). There was a significant correlation between slgE and slgG4 level (r=0.39, p<0.05). These findings suggest that the presence of slgG1 to CRM is response to CRM exposure, and further studies will be needed to evaluate the role of slgG4.

Adolescent↗
PubMed1996

A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes.

A rapid and sensitive dot-blot hybridization assay using in vitro-transcribed digoxigenin-labelled RNA probes (riboprobes) was developed aiming at detection of citrus exocortis viroid (CEVd) in crude sap of infected Citrus medica plants. The protocol includes a very quick and simple preparation of RNA extracts from samples using a denaturation step with formaldehyde. From our results, the employment of this step is highly recommended because the hybridization signals in formaldehyde-denatured samples were significantly stronger when compared with that of extracts without formaldehyde treatment. The assay was found to be sensitive enough to detect 0.1 ng of purified CEVd RNA and was able to detect viroid in 0.2 mg of symptomatic Citrus medica leaves. The use of riboprobes also allowed hybridization under high temperature conditions, avoiding non-specific background.

Digoxigenin↗
PubMed2000

High efficiency genetic transformation of sour orange (Citrus aurantium) and production of transgenic trees containing the coat protein gene of citrus tristeza virus.

In preliminary experiments on Agrobacterium-mediated transformation of citrus, we found transformation events occurring in callus formed from the cambium. Factors affecting Agrobacterium-sour orange (Citrus aurantium L.) interactions, such as culture medium, explant source and culture conditions, were studied to assess competence for transformation in such callus and to improve transformation frequency. Cell divisions and redifferentiation from the transgenic cells leading to transformation events were stimulated more by a combination of benzylaminopurine (BAP) + naphthalene-acetic acid (NAA) in the regeneration-selection medium than by BAP alone. Both age and source of the sour orange plant material affected transformation frequency. Explants from 4-month-old seedlings grown in the greenhouse showed higher transformation frequency than younger and older plant materials, indicating that they had a more suitable balance between dedifferentiated cells competent for transformation and Agrobacterium virulence. Enhancement of transformation frequency enabled us to incorporate the coat protein gene of citrus tristeza virus (CTV) in a sufficient number of sour orange plants to be able to evaluate this strategy for producing CTV-resistant plants.

Journal Article↗
PubMed2005

Volatile constituents of redblush grapefruit (Citrus paradisi) and pummelo (Citrus grandis) peel essential oils from Kenya.

The volatile constituents of cold-pressed peel essential oils of redblush grapefruit (Citrus paradisi Macfadyen forma Redblush) and pummelo (Citrus grandis Osbeck) from the same locality in Kenya were determined by GC and GC-MS. A total of 67 and 52 compounds, amounting to 97.9 and 98.8% of the two oils, respectively, were identified. Monoterpene hydrocarbons constituted 93.3 and 97.5% in the oils, respectively, with limonene (91.1 and 94.8%), alpha-terpinene (1.3 and 1.8%), and alpha-pinene (0.5%) as the main compounds. Sesquiterpene hydrocarbons constituted 0.4% in each oil. The notable compounds were beta-caryophyllene, alpha-cubebene, and (E,E)-alpha-farnesene. Oxygenated compounds constituted 4.2 and 2.0% of the redblush grapefruit and pummelo oils, respectively, out of which carbonyl compounds (2.0 and 1.3%), alcohols (1.4 and 0.3%), and esters (0.7 and 0.4%) were the major groups. Heptyl acetate, octanal, decanal, citronellal, and (Z)-carvone were the main constituents (0.1-0.5%). Perillene, (E)-carveol, and perillyl acetate occurred in the redblush grapefruit but were absent from the pummelo oil. Nootkatone, alpha- and beta-sinensal, methyl-N-methylanthranilate, and (Z,E)-farnesol were prominent in both oils.

Citrus↗
PubMed1996

Cloning and molecular analysis of cDNAs encoding three sucrose phosphate synthase isoforms from a citrus fruit (Citrus unshiu Marc.).

Three partial cDNA clones (pSPS1, pSPS2 and pSPS3) encoding sucrose phosphate synthases (SPS) were isolated by Reverse Transcription (RT)-PCR using first-strand cDNA prepared from the leaf or fruit of citrus (Citrus unshiu Marc.). The nucleotide and deduced amino acid sequences of the three clones showed significant similarities to SPS previously isolated in other plants. A full-length, cDNA clone, CitSPS1, was isolated from a fruit (juice sacs and pulp segment) cDNA library using one (pSPS1) of the three partial clones as a probe. The 3539-bp CitSPS1 clone coded for a 1057-amino acid polypeptide with a predicted molecular mass of 117.8 kDa. The amino acid sequence deduced from the CitSPS1 clone showed homology with SPS from maize (55.8% identity) and spinach (74.0% identity). Genomic Southern blot analysis suggested that CitSPS1 clone represents a lowcopy-number gene. RNA blot analysis of leaf, flower and fruit showed that CitSPS1 and pSPS2 were expressed in all organs. However, the levels of expression of CitSPS1 in young leaves, flowers and immature fruits were low, but high in mature leaves, and fruit. pSPS2 transcripts were barely detectable in young leaves and immature fruits, low in mature leaves, and high in flowers and mature fruits. pSPS3 transcripts were only detected in young and in mature leaves.

Amino Acid Sequence↗
PubMed2002

Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes.

Grapefruit (Citrus paradisi Macf. cv Duncan) plants were transformed with several sequences from citrus tristeza closterovirus (CTV) that varied in terms of position in the CTV genome and virus strain origin in an attempt to obtain resistant plants. The sequences included the capsid protein gene from three different strains, a nontranslatable version of the capsid protein gene, the replicase (RdRp), the minor capsid protein (p27), a highly transcribed gene of unknown function (p20) and the more conserved 3' end of the genomic RNA. Transgenic plants were generated from all of the constructs, except from the p20 and p27 genes. Southern and Western blot analyses demonstrated that stably transformed grapefruit plants were obtained and that at least some transgenes were expressed. In a first effort at virus challenge, 25 transgenic lines were graft inoculated with a severe strain of CTV. Although some transgenic plants averaged lower titers of virus than controls, there was great variability in titer in both controls and transgenic plants, and all were apparently susceptible to the virus.

Blotting, Southern↗
PubMed2004

Introduction of a citrus blight-associated gene into Carrizo citrange [Citrus sinensis (L.) Osbc. x Poncirus trifoliata (L.) Raf.] by Agrobacterium-mediated transformation.

The protein p12 accumulates in leaves of trees with citrus blight (CB), a serious decline of unknown cause. The function of p12 is not known, but sequence analysis indicates it may be related to expansins. In studies to determine the function of p12, sense and antisense constructs were used to make transgenic Carrizo citrange using an Agrobacterium-mediated transformation system. Homogeneous beta-glucuronidase+ (GUS+) sense and antisense transgenic shoots were regenerated using kanamycin as a selective agent. Twenty-five sense and 45 antisense transgenic shoots were in vivo grafted onto Carrizo citrange for further analyses. In addition, 20 sense and 18 antisense shoots were rooted. The homogeneous GUS+ plants contained either the p12 sense or antisense gene (without the intron associated with the gene in untransformed citrus) as shown by PCR and Southern blotting. Northern blots showed the expected RNA in the sense and antisense plants. A protein of identical size and immunoreactivity was observed in seven of nine sense plants but not in nine antisense or non-transgenic plants. At the current stage of growth, there are no visual phenotypic differences between the transgenic and non-transgenic plants. Selected plants will be budded with sweet orange for field evaluation for resistance or susceptibility to CB and general rootstock performance.

Citrus sinensis↗
PubMed2007

Overexpression of citrus polygalacturonase-inhibiting protein in citrus black rot pathogen Alternaria citri.

The rough lemon (Citrus jambhiri) gene encoding polygalacturonase-inhibiting protein (RlemPGIPA) was overexpressed in the pathogenic fungus Alternaria citri. The overexpression mutant AcOPI6 retained the ability to utilize pectin as a sole carbon source, and the overexpression of polygalacturonase-inhibiting protein did not have any effect on the growth of AcOPI6 in potato dextrose and pectin medium. The pathogenicity of AcOPI6 to cause a black rot symptom in citrus fruits was also unchanged. Polygalacturonase-inhibiting protein was secreted together with endopolygalacturonase into culture filtrates of AcOPI6, and oligogalacturonides were digested from polygalacturonic acid by both proteins in the culture filtrates. The reaction mixture containing oligogalacturonides possessed activity for induction of defense-related gene, RlemLOX, in rough lemon leaves.

Alternaria↗
PubMed2004

Estimation of the number of aphids carrying Citrus tristeza virus that visit adult citrus trees.

Aphid species were counted on citrus trees in orchards in Valencia, Spain, in the spring and autumn of 1997, 1998 and 1999. Moericke yellow water traps, the 'sticky shoot' method and counts of established colonies were used in extensive surveys in which 29,502 aphids were recorded and identified. Aphis spiraecola and Aphis gossypii were the most abundant aphid species. The numbers of aphid species landing on mature trees of grapefruit, sweet orange, lemon and clementine and satsuma mandarins, were estimated by counting the numbers of young shoots/tree and aphids trapped on sticky shoots. The proportions of the different aphid species captured were: A. gossypii (53%), A. spiraecola (32%), Toxoptera aurantii (11%), Myzus persicae (1%), Aphis craccivora (1%) and other species (2%). Clementine was the most visited species with 266,700 aphids landing/tree in spring 2000, followed by lemon (147,000), sweet orange (129,150), grapefruit (103,200), and satsuma (92,400). The numbers and relative percentages of aphids carrying Citrus tristeza virus (CTV) were assessed by nested RT-PCR in single closed tubes and analysed by extraction of RNA-CTV targets from trapped aphids. An average of 37,190 CTV-carrying aphids visited each tree in spring 2000 (29 per shoot). The percentage detection of viral RNA in the aphid species that landed were 27% for A. gossypii, 23% for A. spiraecola and 19% for T. aurantii. This high incidence of aphids carrying CTV is consistent with the high prevalence and rapid spread of CTV in sweet orange, clementine, and satsuma mandarins in recent years in the region. The infection rate was proportional to the number of aphids landing/tree.

Animals↗
PubMed1998

Cloning and expression analysis of vacuolar H+-ATPase 69-kDa catalytic subunit cDNA in citrus (Citrus unshiu marc.)1.

To investigate the mechanism of sugar accumulation in fruit vacuoles, a full length cDNA (CitVATP-A) encoding the vacuolar H+-ATPase 69-kDa catalytic subunit was isolated from a cDNA library constructed from citrus fruit (Citrus unshiu Marc.). A 2304-bp insert of CitVATP-A was coded for a 623 amino acid polypeptide with a predicted molecular mass of 68.68 kDa. The deduced amino acid sequence for CitVATP-A showed a 96.5% homology with the carrot homologue. Genomic Southern blot analysis suggested that CitVATP-A is a low-copy number gene. Northern blot analysis of leaves and fruits during the developing stages showed that the level of expression is high in young leaves and is low in mature leaves, and that it increased in both the edible parts and the peel, during fruit growth and maturity.

Amino Acid Sequence↗
PubMed2003

Sodium sulphite yields improved DNA of higher stability for PCR detection of Citrus yellow mosaic virus from citrus leaves.

Citrus yellow mosaic virus (CYMV), a non-enveloped bacilliform DNA virus causes a severe mosaic disease in sweet oranges in India. CYMV is weakly immunogenic, thus serodiagnosis is not a preferred method for its detection. As an alternative a rapid and reliable detection protocol by polymerase chain reaction (PCR) was developed. However, high levels of polyphenolics and tannins in citrus leaves generally interfered with obtaining good quality DNA, and thus affected the reliable detection of virus by PCR. Consequently, we evaluated the addition of sodium sulphite to a DNA extraction protocol used previously and compared the two methods with a commercially available plant DNeasy Kit (Qiagen). The addition of sodium sulphite improved the yield, quality and stability of DNA. The CYMV DNA was not only amplified at lower template DNA concentration, but also provided better DNA yields. In addition, the sodium sulphite extracted DNA survived at various temperatures much longer than those extracted without addition of sodium sulphite or with the commercial kit. The amplified product of CYMV DNA was cloned, sequenced and found to have 89% sequence identity with the only other sequenced Indian isolate of CYMV.

Badnavirus↗
PubMed2001

Identification of IgE-binding components of citrus red mite in sera of patients with citrus red mite-induced asthma.

BACKGROUND: Our previous investigations demonstrated that citrus red mite (CRM) antigen could cause IgE-mediated bronchoconstriction in exposed farmers working on citrus farms. OBJECTIVE: This study was performed to confirm IgE-binding components and major allergens within the CRM antigens. METHODS: Ten subjects who had been diagnosed as having CRM-induced asthma were enrolled. Serum-specific IgE antibodies to CRM antigens were measured by using an ELISA. To identify IgE-binding components and major allergens, SDS-PAGE, 2-dimensional PAGE, IgE-immunoblot analysis, and amino acid sequencing of major allergens were performed. RESULTS: All the asthmatic subjects had high specific IgE antibodies to CRMs. Twelve percent SDS-PAGE analysis showed more than 10 protein bands ranging from 6 to 64 kd. SDS-PAGE and IgE-immunoblot analysis with each individual serum showed 5 IgE-binding components (11, 24, 35, 40, and 64 kd), with 2 (24 and 35 kd) of them bound in more than 50% of the study subjects. Two-dimensional PAGE and IgE-immunoblot analysis demonstrated that the major allergen at 24 kd had 2 bands with different isoelectric points of 4.75 and 5.1. Thirty-five kilodaltons had one band with an isoelectric point of 4.75. All amino acid sequencing of the 2 major allergens was performed, which was not homologous with any previously characterized allergens. CONCLUSION: Five IgE-binding components and 2 major allergens (24 and 35 kd) were identified within the CRM antigen. The N-terminal amino acid sequence of the 2 major allergens (24 and 35 kd) was determined.

Adult↗
PubMed1994

The closely related citrus ringspot and citrus psorosis viruses have particles of novel filamentous morphology.

Some properties of the particles of citrus ringspot virus (CtRSV) and the related citrus psorosis-associated virus (CPsAV) are described. The particles of CtRSV have been reported to be sinuous linear structures about 10 nm in diameter and of two lengths (300 to 500 nm and 1500 to 2500 nm) representing 'top' and 'bottom' sedimentation components. We show that these particles are collapsed double-stranded forms of nucleocapsid-like, highly flexuous open circles formed of filaments 3 to 4 nm in diameter. Top-component filaments had contour lengths of 600 to 1000 nm, i.e. twice that reported for the corresponding collapsed form. Bottom-component filaments had contour lengths about four times longer than those of top-component filaments. The structures suggest that CtRSV represents a new genus (possibly family) related to the tenuiviruses. However, we failed to demonstrate any serological relationship between CtRSV and several tenuiviruses; moreover, the capsid protein sizes and host ranges are quite different. We offer the name Ophiovirus for the proposed new genus.

Antibodies, Viral↗
PubMed1995

The complete nucleotide sequence of RNA 3 of citrus leaf rugose and citrus variegation ilarviruses.

Complete sequence data for the RNA 3 of both citrus leaf rugose (CiLRV) and citrus variegation (CVV) ilarviruses have been determined. The RNAs are 2289 nt (CiLRV) and 2309 nt (CVV) in length and both contain the typical Bromoviridae arrangement of two open reading frames (ORFs) which, when translated, code for proteins that correspond to the Mr 32,000 (32K) putative movement proteins (ORF 1) and the coat proteins (ORF 2) of the respective viruses. The 3' termini of both viruses can be folded to form a secondary structure similar to those reported for other ilarviruses. These are the first complete nucleotide sequences for RNA 3 of members of subgroup 2 of the ilarviruses. The two viruses share substantial homology in nucleic acid sequence, code for identically sized coat proteins and share high levels of identity in the translated products of both ORFs. Although related, these viruses differ sufficiently to be considered distinct. The RNA 3s of CiLRV and CVV appear to be distinct from those of other ilarviruses for which comparable sequence data are available and also from the closely related alfalfa mosaic virus.

Amino Acid Sequence↗
PubMed1992

Studies on natural antioxidants in citrus species. I. Determination of antioxidative activities of citrus fruits.

The antioxidative activities of twenty types of citrus fruits were investigated with a screening method which is based on rat liver microsomal lipid peroxidation induced by dihydronicotinamide adenine dinucleotide phosphate (NADPH) and adenosine diphosphate (ADP). The activities of the exocarp were greater than those of the sarcocarp and the activities from immature fruits (collected in July-August) were greater than those from mature fruits. The strongest antioxidative activity was found in ponkan (Citrus reticulata Blanco) collected in July.

Animals↗
PubMed1991

[Studies on the preparation and evaluation of Kijitsu, the immature citrus fruits. IV. Biological activities of immature fruits of different citrus species].

Biological activities of the 50% ethanolic extract of Kijitsu, dried immature Citrus fruits prepared from C. hassaku Hort. ex Tanaka (H), C. natsudaidai Hayata (N) or allied plants (T, A) were observed. Weak toxicities, weak intestine or uterine relaxant activities, beta-adrenergic activities were shown by in vitro experiments and antiasthmatic activity by intraperitoneal administration. These activities among each extract did not differ. The content of flavonoids (narirutin, naringin hesperidin, neohesperidin) or synephrine did not show much difference among the four dried immature Citrus fruits. These results suggest that H can be used for medicine as the substitute for N, T or A which has been commonly used as Kijitsu. This method is generally applicable to the proof of the biological equivalency of the crude drug and its substitutes.

Adrenergic beta-Agonists↗
PubMed1991

[Pharmacological study on citrus fruits. II. Anti-allergic effect of fruit of Citrus unshiu MARKOVICH (2). On flavonoid components].

A study was carried out to clarify the active component of green fruit of Citrus unshiu MAKOVICH on the type I allergic reaction. The flavonoid component, isolated from its methanolic extract, hesperidin, was found to inhibit histamine release from peritoneal mast cells of rats induced by compound 48/80. Forty eight-hour homologous passive cutaneous anaphylaxis (PCA) in intact rats was significantly inhibited by the oral administration of hesperidin. However, the anti-allergic action on PCA was not observed in adrenalectomized rats. These results suggests that hesperidin is an effective component of the fruit of Citrus unshiu with the anti-allergic action against the type I reaction.

Adrenalectomy↗
PubMed2001

Volatile flavor components of ripe and overripe ki-mikans (Citrus flaviculpus Hort. ex Tanaka) in comparison with Hyuganatsu (Citrus tamurana Hort. ex Tanaka).

The volatile flavor components of ripe and overripe ki-mikan (Citrus flaviculpus Hort. ex Tanaka) peel oil samples, which had been isolated by cold-pressing, were investigated by capillary GC and GC-MS, and compared with the Hyuganatsu (Citrus tamurana Hort. ex Tanaka) flavor. Limonene (ripe fruit, 82.44%; overripe fruit, 73.10%) was the most abundant compound in the ki-mikan oil, this being followed by gamma-terpinene (8.83% and 13.74%), trans-beta-farnesene (1.76% and 3.12%) and myrcene (1.54% and 1.13%). The composition of overripe ki-mikan oil was characterized by higher amounts of aliphatic and sesquiterpene hydrocarbons, monoterpene and sesquiterpene alcohols, ketones and esters than that of ripe ki-mikan oil. Monoterpene hydrocarbons, especially limonene (84.78%), were predominant in Hyuganatsu oil. The CPO composition of ki-mikan was qualitatively similar to that of Hyuganatsu, but differed quantitatively. The content of sesquiterpene hydrocarbons was higher in the ki-mikan oil samples than in Hyuganatsu oil, while ketones showed the opposite predominance. These differences were more evident in the trans-beta-farnesene and l-carvone contents. The ratio of both these compounds could be used to distinguish ki-mikan oil from Hyuganatsu oil.

Citrus↗