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Detection of Citrus psorosis virus in the northwestern citrus production area of Argentina by using an improved TAS-ELISA.

Citrus Psorosis in Argentina is a serious disease. Citrus is produced in two regions located in the northeast (NE) and northwest (NW) area of the country. These two areas have different climates and soil types, and therefore different citrus species and varieties are cultivated. In the NE region, Psorosis is epidemic, and in the NW region, the disease was described on several occasions since 1938, but it is not observed commonly in the orchards. Recently, trees with symptoms of Psorosis were observed in the Tucumán and Salta Provinces located in the NW region. Epidemiological studies in Argentina and Texas suggested that the disease is spread naturally by an unknown vector. The causal agent of the disease is the Citrus psorosis virus (CPsV), which can be detected by TAS-ELISA, RT-PCR and indicator plants. A new more rapid TAS-ELISA-HRP (horseradish peroxidase) is described which is more reliable, faster and more sensitive than the currently used for this virus, the TAS-ELISA-AP (alkaline phosphatase). Psorosis was detected by this improved method in few trees in the orchards of the Tucumán Province, in the NW citrus region, although natural spread does not seem to occur.

Alkaline Phosphatase↗

Progress on strain differentiation of Citrus tristeza virus and its application to the epidemiology of citrus tristeza disease.

Citrus tristeza virus (CTV) occurs in most citrus producing regions of the world, and it is the most serious viral pathogen of citrus. With the recent establishment of the brown citrus aphid, Toxoptera citricida, its most efficient vector, on Madeira Island (Portugal) and in Florida (USA) and the countries of the Caribbean Basin, the impact of CTV is likely to increase in these regions. Since there are many strains of CTV and CTV infections frequently occur as mixtures of several strains, it is necessary to be able to distinguish the strains for regulatory purposes, disease management and epidemiology. We describe the evolution of techniques developed to detect CTV and to differentiate the individual strains, and present the results of tests using these latest methods on CTV isolates from mainland Portugal, Madeira Island and Florida. Mild and decline-inducing strains of CTV were detected in mainland Portugal and mild, decline-inducing and severe stem pitting strains on Madeira Island. In Florida we demonstrated the presence of infections that reacted with probes made against stem pitting strains not previously detected there. It is concluded that CTV presents a significant threat to citrus production in mainland Portugal, on Madeira Island and in the neighbouring countries of the Mediterranean Basin, as well as in Florida, elsewhere in the USA and throughout the Caribbean Basin, especially following the widespread establishment of T. citricida throughout the region.

Amino Acid Sequence↗

Identification of two chilling-regulated 1-aminocyclopropane-1-carboxylate synthase genes from citrus (Citrus sinensis Osbeck) fruit.

Diurnal change in the temperature below or above 12.5 degrees C hastens the degreening of citrus peel and elicits the phytohormone ethylene production in citrus fruit. Ethylene triggers the degradation of chlorophyll and synthesis of carotenoids in citrus peel. To investigate if ethylene is required for the degreening of citrus peel elicited by low temperatures, we studied the chilling-regulated gene expression of ACC synthase, one of the key enzymes catalyzing ethylene biosynthesis. We isolated and characterized a chilling-inducible 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) gene, CS-ACS1, and a chilling-repressible gene, CS-ACS2, from citrus peel. The CS-ACS1 transcript 1.7 kb in length encodes a polypeptide of 483 amino acids (Mr 54,115, pI 6.63), whereas the CS-ACS2 transcript of 1.8 kb encodes a polypeptide of 477 amino acids (Mr 53,291, pI 6.72). Both genes showed a rapid but transient induction (within 2.4 h) of transcripts upon rewarming after the chilling (4 degrees C) treatment. After 24 h of incubation at room temperature, CS-ACS1 mRNA diminished to an undetectable level, whereas the CS-ACS2 mRNA regained its basal level of expression attained prior to the chilling treatment. Chilling-induced ethylene production and ACC accumulation were also observed upon rewarming. Both genes were also induced by the wound stress (excision). The protein synthesis inhibitor cycloheximide super-enhances the accumulation of both ACS transcripts at room temperature. Molecular analysis of the 3.3 kb genomic DNA of CS-ACS1 revealed that this gene consists of three introns and four exons. The intron 3 is exceptionally large ( 1.2 kb) and shares significant homology with mitochondrial DNA, supporting the intron-late theory.

Amino Acid Sequence↗

[Phosphorus translocation and distribution in intercropping systems of soybean (Glycine max) and citrus (Citrus poonensis)].

A field mini-plot experiment was conducted on clay loamy oxisol using 32P trace technique when P fertilizer was applied in three depth soil (15, 35 and 55 cm soil layer) to compare P absorption, distribution and translocation in plant organ and soil profile under soybean and citrus monoculture and intercropping at Taoyuan Experimental Station of Agroecosystem Research of Chinese Academy of Science. Total P uptake (PT) and P accumulation in different parts (PA) of soybean were remarkably decreased under intercropping. When 32P was applied in topsoil (15 cm soil layer), 32P uptake (32PT) by soybean was significantly lower in intercropping than in monoculture. Whereas 32PT uptake by soybean was significantly greater in intercropping than in monoculture when 32P was applied in deep soil layer (35 cm or 55 cm soil layer). However, considerable difference was not observed for 32P translocation and distribution among soybean organs. 32PT uptake by citrus was much lower under intercropping than under monoculture. The P uptake by citrus newly could be transferred rapidly to aboveground and prior to active growing organ. Intercropping did not affect 32P distribution in citrus organ, but when P was applied in deep soil layer, the speed of 32P transferred to aboveground and active organ was slowed down. P mobility was strengthened in soil profile, and P of deep soil layer was promoted to move to topsoil in intercropping. The experimental results showed the optimal depth of applied P should be within 20 cm soil layer in soybean-citrus intercropping system.

Biomass↗

Enumerative and binomial sampling plans for citrus mealybug (Homoptera: pseudococcidae) in citrus groves.

The spatial distribution of the citrus mealybug, Planococcus citri (Risso) (Homoptera: Pseudococcidae), was studied in citrus groves in northeastern Spain. Constant precision sampling plans were designed for all developmental stages of citrus mealybug under the fruit calyx, for late stages on fruit, and for females on trunks and main branches; more than 66, 286, and 101 data sets, respectively, were collected from nine commercial fields during 1992-1998. Dispersion parameters were determined using Taylor's power law, giving aggregated spatial patterns for citrus mealybug populations in three locations of the tree sampled. A significant relationship between the number of insects per organ and the percentage of occupied organs was established using either Wilson and Room's binomial model or Kono and Sugino's empirical formula. Constant precision (E = 0.25) sampling plans (i.e., enumerative plans) for estimating mean densities were developed using Green's equation and the two binomial models. For making management decisions, enumerative counts may be less labor-intensive than binomial sampling. Therefore, we recommend enumerative sampling plans for the use in an integrated pest management program in citrus. Required sample sizes for the range of population densities near current management thresholds, in the three plant locations calyx, fruit, and trunk were 50, 110-330, and 30, respectively. Binomial sampling, especially the empirical model, required a higher sample size to achieve equivalent levels of precision.

Animals↗

Interaction between endophytic bacteria from citrus plants and the phytopathogenic bacteria Xylella fastidiosa, causal agent of citrus-variegated chlorosis.

AIMS: To isolate endophytic bacteria and Xylella fastidiosa and also to evaluate whether the bacterial endophyte community contributes to citrus-variegated chlorosis (CVC) status in sweet orange (Citrus sinensis [L.] Osbeck cv. Pera). METHODS AND RESULTS: The presence of Xylella fastidiosa and the population diversity of culturable endophytic bacteria in the leaves and branches of healthy, CVC-asymptomatic and CVC-symptomatic sweet orange plants and in tangerine (Citrus reticulata cv. Blanco) plants were assessed, and the in vitro interaction between endophytic bacteria and X. fastidiosa was investigated. There were significant differences in endophyte incidence between leaves and branches, and among healthy, CVC-asymptomatic and CVC-symptomatic plants. Bacteria identified as belonging to the genus Methylobacterium were isolated only from branches, mainly from those sampled from healthy and diseased plants, from which were also isolated X. fastidiosa. CONCLUSIONS: The in vitro interaction experiments indicated that the growth of X. fastidiosa was stimulated by endophytic Methylobacterium extorquens and inhibited by endophytic Curtobacterium flaccumfaciens. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the first evidence of an interaction between citrus endophytic bacteria and X. fastidiosa and suggests a promising approach that can be used to better understand CVC disease.

Actinomycetales↗

Amplification of Citrus tristeza virus from a cDNA clone and infection of citrus trees.

Isolates of the Closterovirus, Citrus tristeza virus (CTV), are populations of disparate genotypes and defective RNAs developed during long periods of vegetative propagation of citrus trees. Because it has not been possible to obtain pure cultures of the virus, it is not known what components of the population are primarily responsible for induction of diseases. We previously developed an infectious cDNA clone from which in vitro-produced RNA transcripts could infect protoplasts (Satyanarayana et al., 1999, Proc. Natl. Acad. Sci. USA 96, 7433-7438). However, neither the RNA transcripts nor virions from transcript-infected protoplasts were competent for infection of citrus trees. Using a green fluorescent protein-marked virus as inoculum, we found that the approximately 20-kb RNA from virions or transcripts of cDNA infected only a small percentage of protoplasts ( approximately 0.01%), but virions could infect more than 80% of the protoplasts. Based on this information, we amplified the virus from the cDNA clone (recombinant virus) by successive passages in protoplasts using virions in crude sap as inoculum. By the third to seventh passages in protoplasts, maximal amounts of recombinant progeny virus were produced, which were used for inoculation of small citrus trees by slashing stems in the presence of virion preparations. A relatively high percentage of plants became infected with the recombinant virus from protoplasts, resulting in the first defined pure culture of CTV in plants. The comparative biology of the pure culture of recombinant CTV with that of the parental population in planta demonstrated that the recombinant virus retained through all of the recombinant DNA manipulations the normal functions of replication, movement, and aphid transmissibility, and had a symptom phenotype indistinguishable from that of the parental population. Additionally, fulfilling Koch's postulates of the first pure culture of CTV in plants suggested that the major genotype of the CTV T36 population is the primary determinant of the symptom phenotype. We could distinguish no biological contributions resulting from the minor genotypes and defective RNAs of the parental population.

Animals↗

Regulation of gibberellin 20-oxidase gene expression and gibberellin content in citrus by temperature and citrus exocortis viroid.

A cDNA clone coding for a gibberellin (GA) 20-oxidase ( CcGA20ox1), an enzyme of GA biosynthesis, which when expressed in vitro catalyzed the conversion of GA(12) to GA(9) and of GA(53) to GA(20), was isolated from the citrus hybrid Carrizo citrange (C itrus sinensis x Poncirus trifoliata). Transcripts of CcGA20ox1 were abundant in the apex and leaves and much less abundant in internodes, nodes and roots. Seedlings of Carrizo citrange cultured under a 32 degrees C/27 degrees C (day/night) regime elongated more than seedlings growing under 17 degrees C/12 degrees C conditions. The effect of higher temperature was associated with more CcGA20ox1 transcripts and with higher content of GA(1), the main active GA in citrus, in the shoot. The infection of Etrog citron ( Citrus medica) plants with citrus exocortis viroid (CEVd), which produces a stunted phenotype, reduced the levels of transcripts in the apical shoot hybridizing to the gene CcGA20ox1 of Carrizo citrange and the content of GA(1). Thus GA(1) content correlated with CcGA20ox1 transcript levels. In contrast, results for gibberellic acid (GA(3)) and paclobutrazol applications to Carrizo citrange showed that CcGA20ox1 expression was subject to feed-back regulation. These observations indicate that the feed-back regulation of GA20ox operates mostly when the levels of active GAs have been dramatically altered. The results also show that the growth reduction induced by environmental (temperature) and biotic (CEVd) factors may be partially due to the modulation of the expression of GA20ox genes.

Citrus↗

New occupational allergen in citrus farmers: citrus red mite (Panonychus citri).

BACKGROUND: There have been several reports of occupational allergy to spider mites (Tetranychidae), but no published report has described citrus red mite (CRM, Panonychus citri)-induced occupational asthma confirmed by specific bronchial challenge. OBJECTIVE: The purpose of this study was to evaluate clinical and immunologic characteristics of CRM-induced occupational asthma. METHODS AND RESULTS: We encountered 16 cases of CRM-induced occupational asthma among farmers cultivating citrus fruits. Asthmatic attacks corresponded closely with their work on citrus farms. The mean duration of the latent period was 12.9 (range 7 to 20) years. During their first visit to our clinic, nine patients with FEV1 lower than 70% of predictive value showed reversible airway obstruction after inhalation of bronchodilator, and seven with FEV1 greater than 70% of predictive value showed airway hyperresponsiveness to methacholine. Fifteen of the 16 also complained of recurrent nasal symptoms, which had developed at an earlier time than the asthmatic symptoms. They showed strong positive reactions to CRM extract on skin prick test (A/H ratio > or = 1.0) and had high serum specific IgE antibody against CRM which was detected by ELISA. Skin prick test with common inhalant allergens revealed that 10 had an isolated positive response to CRM with negative results to common inhalant allergens in their environment. The ELISA inhibition tests with CRM demonstrated significant inhibitions by CRM in a dose-dependent manner, while minimal inhibitions were noted by D. pteronyssinus and mugwort allergens. CONCLUSION: These findings suggest that CRM could induce IgE-mediated bronchoconstriction in exposed workers on citrus farm.

Adult↗

Citrus fruit bitter flavors: isolation and functional characterization of the gene Cm1,2RhaT encoding a 1,2 rhamnosyltransferase, a key enzyme in the biosynthesis of the bitter flavonoids of citrus.

Species of the genus Citrus accumulate large quantities of flavanones that affect fruit flavor and have been documented to benefit human health. Bitter species, such as grapefruit and pummelo, accumulate bitter flavanone-7-O-neohesperidosides responsible, in part, for their characteristic taste. Non-bitter species, such as mandarin and orange, accumulate only tasteless flavanone-7-O-rutinosides. The key flavor-determining step of citrus flavanone-glycoside biosynthesis is catalyzed by rhamnosyltransferases; 1,2 rhamnosyltransferases (1,2RhaT) catalyze biosynthesis of the bitter neohesperidosides, while 1,6 rhamnosyltransferases (1,6RhaT) catalyze biosynthesis of the tasteless rutinosides. We report on the isolation and functional characterization of the gene Cm1,2RhaT from pummelo which encodes a citrus 1,2RhaT. Functional analysis of Cm1,2RhaT recombinant enzyme was conducted by biotransformation of the substrates using transgenic plant cell culture. Flavanones and flavones, but not flavonols, were biotransformed into 7-O-neohesperidosides by the transgenic BY2 tobacco cells expressing recombinant Cm1,2RhaT. Immunoblot analysis established that 1,2RhaT protein was expressed only in the bitter citrus species and that 1,6RhaT enzyme, whose activity was previously documented in non-bitter species, was not cross-reactive. Expression of Cm1,2RhaT at the RNA level was prominent in young fruit and leaves, but low in the corresponding mature tissue, thus correlating well with the developmental pattern of accumulation of flavanone-neohesperidosides previously established. Phylogenetic analysis of the flavonoid glycosyltransferase gene family places Cm1,2RhaT on a separate gene cluster together with the only other functionally characterized flavonoid-glucoside rhamnosyltransferase gene, suggesting a common evolutionary origin for rhamnosyltransferases specializing in glycosylation of the sugar moieties of flavonoid glucosides.

Amino Acid Sequence↗

In vitro fermentation of cellulose, beet pulp, citrus pulp, and citrus pectin using fecal inoculum from cats, dogs, horses, humans, and pigs and ruminal fluid from cattle.

We evaluated the influence of gastrointestinal tract microflora from several species on fiber fermentation characteristics in vitro. Selected fibrous substrates (cellulose, beet pulp, citrus pulp, and citrus pectin) were incubated for 6, 12, 24, and 48 h with ruminal fluid from cattle or feces from dogs, cats, pigs, horses, or humans. When data were pooled across all substrates and fermentation times, OM disappearance (29.4%) and acetate, propionate, butyrate, and total short-chain fatty acid (SCFA) production (1.09, .41, .12, and 1.61 mmol/g of OM, respectively) were lowest (P < .05), and lactate production (.23 mmol/g of OM) was greatest (P < .05) for horse fecal microflora compared with samples from the other species. The greatest (P < .05) acetate production resulted when substrates were fermented by cat fecal microflora (2.38 mmol/g of OM). The greatest (P < .05) propionate productions resulted from pig fecal and cattle ruminal microflora (.88 and .83 mmol/g of OM, respectively), and the greatest (P < .05) butyrate productions resulted from human and pig fecal microflora (.39 and .40 mmol/g of OM, respectively). Total SCFA production was greatest (P < .05) for cat fecal microflora (3.38 mmol/g of OM). When data were pooled across the species, substrate OM disappearance and SCFA production ranked from least to greatest in the following order: cellulose < beet pulp < citrus pulp < citrus pectin. The fermentability of different fibrous substrates by fecal or ruminal microflora from various species seems to be dependent not only on the fermentative activity of the microbial population but on other factors as well, perhaps lag time and rate of digesta passage.

Adult↗

Electrophoretic and immunological evidence of unique proteins in leaves of citrus trees: application to citrus blight detection.

From the leaf tissue of healthy and blighted citrus trees 10-30 kDa soluble fractions were compared to find biochemical markers of tissue in the disease state. Using a non-denaturing extraction technique coupled with ultrafiltration, a resulting 10-30 kDa healthy and citrus blight fraction was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two distinct and adjacent bands for blight at an Mr of approximately 12,500 were separated. These bands were visible with Coomassie Brilliant Blue and silver stain but were negative to glycoprotein stains. An antiserum prepared against proteins isolated by preparative electrophoresis reacted only with the blight fractions and was distinctly different from healthy fractions when Western blotted. Only the gel region (Mr 12,500-13,000) of citrus blight sources was positive to the antiserum when compared with disease and nondisease stress sources. Results indicate that identification of specific proteins may be a way to diagnose the onset of citrus blight prior to visible tree symptoms.

Biomarkers↗

A population of variants of a viroid closely related to citrus viroid-I in citrus plants.

Sequencing analyses showed a population of variants consisting of 325-330 nucleotides (nt) of a viroid closely related to citrus viroid (CVd)-I in citrus plants. These variants, for which we propose the tentative acronym CVd-I-LSS (low sequence similarity), have only 82-85% sequence similarities to CVd-I variants. A phylogenetic tree showed that the CVd-I-LSS variants formed an individual cluster that was distinct from that of the CVd-I variants, but no intermediate variants were found which could continuously connect the population of CVd-I-LSS variants with that of CVd-I variants. Citrons (Citrus medica L.) inoculated with the CVd-I-LSS seemed to show not only moderate leaf bending like citrons infected with CVd-I but also slightly more severe epinasty than citrons with CVd-I. Other biological properties of CVd-I-LSS, such as the host range, will need to be determined in order to clarify whether CVd-I-LSS is a new viroid species or a distinct strain of CVd-I. Most of the nucleotide changes between the CVd-I-LSS and CVd-I variants were found at complementary positions of the upper and lower strands within the putative pathogenic, variable, and right terminal domains, as well as in the region surrounding the central conserved region of the predicted secondary structure of CVd-I-LSS.

Base Sequence↗

Phenols in citrus peel byproducts. Concentrations of hydroxycinnamates and polymethoxylated flavones in citrus peel molasses.

In addition to the main flavanone glycosides (i.e., hesperidin and naringin) in citrus peel, polymethoxylated flavones and numerous hydroxycinnamates also occur and are major phenolic constituents of the molasses byproduct generated from fruit processing. Although a small number of the hydroxycinnamates in citrus occur as amides, most occur as esters and are susceptible to alkaline hydrolysis. This susceptibility to alkaline hydrolysis was used in measuring the concentrations of hydroxycinnamates in citrus peel molasses. The highest concentrations of hydroxycinnamates occurred in molasses of orange [C. sinensis (L.) Osbeck] and tangerine (C. reticulata Blanco.) compared to grapefruit (C. paradisi Macf.) and lemon [C. limon (L.) Burm.]. Concentrations of two phenolic glucosides, phlorin (phloroglucinol-beta-O-glucoside) and coniferin (coniferyl alcohol-4-beta-O-glucoside), were also measured. Measurements of the polymethoxylated flavones in molasses from several tangerine and orange varieties showed that these compounds occurred in the highest amounts in Dancy tangerine, whereas samples from two other tangerine molasses contained significantly lower levels, similar to those in the molasses samples from late- and early/mid-season oranges.

Chromatography, High Pressure Liquid↗

Essential oil composition of Citrus meyerii Y. Tan. and Citrus medica L. cv. Diamante and their lemon hybrids.

In this paper we report the volatile fraction composition of Citrus meyerii Y. Tan. and Citrus medica L. cv. Diamante and two new lemon hybrids obtained by cross-breeding them with the tetraploid Citrus limon Burm. cv. Cavone. Both parent and hybrid oils were laboratory-extracted from the peel fruits and analyzed by HRGC-MS and HRGC-FID. Sixty-three components were fully characterized by mass spectra, linear retention indices, and injection of standards. The average composition as single components for all the oils analyzed is reported. Moreover, the data obtained were statistically analyzed. Since limonene is by far the main component of all the essential oils examined, analysis of variance and multivariate analysis gave interesting information on the similarities and differences between the oils analyzed. The new hybrid oils analyzed have potential commercial value because they could be an acceptable alternative to the valuable lemon oil.

Chromatography, Gas↗

Acute intoxication of cyclosporin caused by coadministration of decoctions of the fruits of Citrus aurantium and the Pericarps of Citrus grandis.

In order to investigate the effects of Citrus herbs on cyclosporin absorption and disposition, swine were given cyclosporin (10 mg/kg) with or without decoctions of Citri Aurantii Fructus (CAF) or Citri Grandis Pericarpium (CGP) in a crossover design. FPIA method (fluorescence polarization immunoassay) was used to determine the blood concentration of cyclosporin. The decoctions were characterized by their flavanone contents. Our results indicated that the coadministration of CAF and CGP significantly increased the Cmax of cyclosporin by 64% and 79%, respectively. The AUC of cyclosporin was significantly elevated by 97% when coadministered with CGP. Among the swine, 1/5 and 3/5 exhibited acute toxicity of cyclosporin after concomitant intake of CAF and CGP, respectively. This indicates an interaction of Citrus compounds with a commonly used drug. We suggest when cyclosporin is coadministered with these Citrus decoctions, the blood concentration of cyclosporin should be carefully monitored for dose adjustment to avoid cyclosporin intoxication.

Animals↗

Viral-like symptoms induced by the ectopic expression of the p23 gene of Citrus tristeza virus are citrus specific and do not correlate with the pathogenicity of the virus strain.

Ectopic expression of the p23 gene from a severe (T36) strain of Citrus tristeza virus (CTV) induces viral-like symptoms in Mexican lime. Here, we report that expressing the same gene from a mild strain induced similar symptoms that correlated with accumulation of p23 protein irrespective of the source strain. CTV inoculation of transgenic limes showing CTV-like leaf symptoms and high p23 accumulation did not modify symptoms initially, with the virus titer being as in inoculated nontransgenic controls; however, at later stages, symptoms became attenuated. Transformation with p23-T36 of CTV-susceptible sweet and sour orange and CTV-resistant trifoliate orange also led to CTV-like leaf symptoms that did not develop when plants were transformed with a truncated p23 version. In transgenic citrus species and relatives other than Mexican lime, p23 was barely detectable, although symptom intensity correlated with levels of p23 transcripts. The lower accumulation of p23 in sweet and sour orange compared with Mexican lime also was observed in nontransgenic plants inoculated with CTV, suggesting that minimal p23 levels cause deleterious effects in the first two species. Conversely, transgenic expression of p23 in CTV nonhost Nicotiana spp. led to accumulation of p23 without phenotypic aberrations, indicating that p23 interferes with plant development only in citrus species and relatives.

Amino Acid Sequence↗

[Pharmacological study on citrus fruits. I. Anti-allergic effect of fruit of Citrus unshiu Markovich (1)].

A study was carried out to examine the effect of 50% ethanolic extract from green fruit of Citrus unshiu Markovich on type I, II and IV allergic reactions. Forty eight-hour homologous passive cutaneous anaphylaxis (PCA) in rats as a typical model of the type I reaction was significantly inhibited by the oral administration of the extract. Complement-dependent cytolysis of sheep red blood cell as a typical model of the type II reaction was significantly inhibited by incubation with it. Contact dermatitis in mice as a model of the type IV reaction caused by picryl chloride or sheep red blood cell was significantly inhibited by the oral application of it. The anti-allergic actions decreased with the growth of fruit of Citrus unshiu. These results suggested that the 50% ethanolic extract from green fruit of Citrus unshiu have anti-allergic actions against the type I, II and IV reactions.

Animals↗