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RAPD profile and antibiotic susceptibility of Xylella fastidiosa, causal agent of citrus variegated chlorosis.

AIMS: The aim of this study was to evaluate the diversity of Xylella fastidiosa isolated from citrus trees affected by Citrus Variegated Chlorosis (CVC). METHODS AND RESULTS: The antibiotic susceptibility by agar disc diffusion and minimum inhibitory concentration (MIC) methods was observed for all drug evaluated, except for penicillin-G. Genetic diversity by RAPD analysis revealed three major groups (citrus, coffee and grapevine), being the citrus group more similar with the coffee group than with the grapevine group. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the possibility to use these antibiotics susceptibility as markers in the development of a cloning vector and penicillin-G could be used as a selective marker for the isolation of X. fastidiosa from citrus plants.

Anti-Bacterial Agents↗

Monitoring study of urinary metabolites and selected symptomatology among Florida citrus workers.

A cross-sectional monitoring study was made of citrus fieldworkers employed during an entire citrus growing season in Florida. A survey questionnaire was administered to 1811 fieldworkers employed as applicators, mixers, loaders, tractor drivers, general combination workers, and pickers on 436 citrus groves throughout Florida. The study was designed to evaluate exposure to organophosphorus pesticides by analyzing the urine of citrus fieldworkers for dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP), dimethyl dithiophosphate (DMDTP), diethyl phosphate (DEP), diethyl thiophosphate (DEPT), and diethyl dithiophosphate (DEDTP), and to relate the findings to the reported occurrence of selected health symptoms associated with pesticide intoxication. Urine samples were collected for analysis from 332 spray-season workers and 265 harvest-season workers. Measurable DEP residue values were found in 68% of the spray season workers and 43% of the harvesters. However, with the exception of DEP and DETP values in the urine of spray-season workers, metabolite residues were low, often at, or just above, detection limits (which were usually 0.02 ppm but occasionally rose higher, with a few at 0.03 ppm and a very few at 0.04 ppm). Although there was clearly exposure to organophosphates among the citrus fieldworkers, there was no apparent association between the reported health symptoms and the relatively low levels of organophosphate metabolites found in the urine of the workers.

Agricultural Workers' Diseases↗

Effect of dietary wheat bran and dehydrated citrus fiber on 3,2'-dimethyl-4-aminobiphenyl-induced intestinal carcinogenesis in F344 rats.

The effect of dietary wheat bran and dehydrated citrus fiber on 3,2'-dimethyl-4-aminobiphenyl (DMAB)-induced colon and small intestinal carcinogenesis was studied in male F344 rats. Weanling rats were fed semipurified diets containing 5% alphacel, 5% alphacel + 15% wheat bran or 5% alphacel + 15% citrus fiber. At 7 weeks of age, all animals, except vehicle-treated controls, received weekly s.c. injections of 50 mg DMAB/kg body weight for 20 weeks. The DMAB- or vehicle-treated groups were autopsied 20 weeks after the last injection of DMAB. The animals fed the wheat bran diet and treated with DMAB had a lower incidence (number of animals with tumors) and multiplicity (number of tumors/tumor-bearing animal) of colon and small intestinal tumors than did those fed the control diet and treated with DMAB. Animals fed the diet containing citrus fiber developed fewer small intestinal tumors (incidence and multiplicity) than did the rats fed the control diet; the number of adenocarcinomas was reduced in rats fed the citrus fiber diet. This study thus indicates that diets containing wheat bran and citrus fiber reduce the risk for DMAB-induced intestinal cancer and that the protection against colon cancer depends on the type of fiber.

Aminobiphenyl Compounds↗

Nitrate improves growth in salt-stressed citrus seedlings through effects on photosynthetic activity and chloride accumulation.

We analyzed the effects of nitrate availability on growth of Navelina (Citrus sinensis (L.) Osbeck) scions grafted on three citrus rootstocks differing in salt tolerance: Carrizo citrange (Citrus sinensis (L.) Osbeck x Poncirus trifoliata (L.) Raf.), Citrus macrophylla Wester and Cleopatra mandarin (Citrus reshni Hort. ex Tanaka). Salt stress reduced total plant biomass by 27-38%, whereas potassium nitrate supplementation partially counteracted this effect by increasing dry matter and new leaf area. Salinized Carrizo citrange had the greatest response to nitrate supplementation, whereas the effects on salinized Cleopatra mandarin and C. macrophylla were less apparent. Nitrogen and chlorophyll contents and photosynthetic activity also increased in leaves of the nitrate-supplemented salinized plants. In salinized plants, nitrate supplementation reduced leaf abscission, stimulated photosynthetic activity and increased growth of new leaves. The nitrate treatment did not modify chloride concentration in leaves, but it reduced chloride concentrations in Carrizo and Macrophylla roots. Therefore, in both rootstocks, chloride content was similar in mature leaves, higher in immature leaves and lower in roots of the nitrate-supplemented salinized plants compared with salinized plants unsupplemented with nitrate. We suggest that the nitrate-induced stimulation of growth reduced chloride concentration in roots through the reallocation of chloride to new leaves.

Chlorides↗

Genetic analysis of hrp-related DNA sequences of Xanthomonas campestris strains causing diseases of citrus.

The hrp gene cluster of strains of Xanthomonas campestris that cause diseases of citrus was examined by Southern hybridization of genomic DNA and by restriction endonuclease analysis of enzymatically amplified DNA fragments of the hrp gene cluster. The hrp genes were present in all strains of the pathovars of X. campestris tested in this study, including strains of the three aggressiveness groups of the citrus bacterial spot pathogen, X. campestris pv. citrumelo. X. campestris pv. citri strains in groups A, B, and C, which cause citrus canker A, B, and C, respectively, each produced characteristic restriction banding patterns of amplified hrp fragments. The restriction banding patterns of all strains within each group were identical. In contrast, restriction fragment length polymorphism was evident among strains of the moderately and weakly aggressive groups of X. campestris pv. citrumelo. X. campestris pv. citrumelo strains in the highly aggressive group had a homogeneous restriction banding pattern. The characteristic banding patterns obtained for each bacterial group indicate that X. campestris strains causing disease in citrus can be reliably differentiated and identified by restriction analysis of amplified DNA fragments of the hrp gene cluster. In addition, the phylogenetic analysis based on the restriction banding patterns of amplified fragments suggests a polyphyletic relationship of the hrp genes among the strains of X. campestris that cause disease in citrus.

Citrus↗

Radioprotective effects of citrus extract against gamma-irradiation in mouse bone marrow cells.

The radioprotective effects of citrus extract were investigated by using the micronucleus test for anticlastogenic and cell proliferation activity. A single intraperitoneal (i.p.) injection of citrus extract (Citrus aurantium var. amara) at 250, 500, 1000 mg/kg body weight 1 h prior to gamma-ray irradiation (1.5 Gy) reduced the frequencies of micronucleated polychromatic erythrocytes (MnPCE(S)) and normochromatic erythrocytes (MnNCE (S)). All three doses of citrus extract significantly reduced the frequencies of MnPCEs and MnNCEs in mice bone marrow compared to non-drug-treated irradiated control (p < 0.005-0.05). The optimum dose for protection in mouse was 250 mg/kg to protect mice bone marrow 2.2-fold against the side effects of gamma-irradiation with respect to the non-drug-treated irradiated control. The flavonoids were contained in citrus extract, probably to show protective activity, and reduced the clastogenic effect of radiation on mice bone marrow. Therefore fruits and vegetables contain flavonoids to be useful as protective effects under such stress conditions as irradiation.

Animals↗

Undesirable effects of citrus juice on the pharmacokinetics of drugs: focus on recent studies.

It is well known that intake of grapefruit juice affects the pharmacokinetics of various kinds of drugs. It has been reported that other citrus juices also interact with certain drugs. To re-evaluate citrus juice-drug interactions based on currently available evidence, a literature search was conducted for new and updated information since the grapefruit juice-drug interaction was last reviewed in 1998. MEDLINE (1998-October 2004) was accessed and more than 200 reports were found. The effects of grapefruit juice ingestion on the pharmacokinetics of orally administered drugs have been reported for 40 drugs since the reviews published in 1998. Increases in either area under the concentration-time curve (AUC) or maximum plasma concentration (C(max)) were found with 34 of these, the major mechanism being considered to be inactivation of intestinal cytochrome P450 3A4, a so-called mechanism-based inhibition. Although recent reports point to the inhibitory effects of grapefruit juice on the function of P-glycoprotein, which transports substrates from enterocytes back into the lumen, the contribution to the bioavailability of drugs that are substrates of P-glycoprotein has not been established yet. Dramatic decreases in AUC and C(max) for two drugs in association with grapefruit juice ingestion has been reported and, in these cases, inhibitory effects on organic anion transporting polypeptide, which mediates absorption from the intestinal lumen to enterocytes, might be involved. Other citrus juices such as Seville (sour) orange juice and commonly ingested varieties of orange juice also showed significant effects on the AUC and C(max) of some drugs. Although the situation is complex and uncertainties remain, we recommend that patients avoid citrus juice intake while taking medications and that healthcare providers advise against citrus juice intake in this setting until any interactions with subject drugs can be clarified in clinical studies.

ATP Binding Cassette Transporter, Subfamily B↗

Effects of citrus pulp in high urea rations for steers.

Effects of pelleted and conventional citrus pulp as a replacement for corn, with soybean meal added to keep protein comparable, were tested in rations with 5% urea and 33.33% sugarcane bagasse for fistulated steers. Thus, all rations were low in readily fermented carbohydrates other than those of corn or citrus pulp. Evaluation criteria were concentrations of urea in blood and of pH, ammonia, and volatile fatty acids of rumen fluid. Citrus pulp for diets 1, 2, 3, and 4 was 0, 19, 38, or 55%. Rumen fluid and blood were sampled 1 h before and 2, 4, 7, and 12 h after feed was placed directly into the rumen. No differences between pelleted and conventional pulp or among time trends were significant except that for both forms rumen ammonia was lower with the two highest percents of citrus pulp. Addition of citrus pulp at 0, 19, 38, or 55% of the ration reduced rumen pH (6.85, 6.65, 6.61, 6.51). Blood urea and rumen ammonia decreased in steers fed 19, 38, or 55% pulp; thus, the acetic to propionic ratio was higher. Butyric acid changed only in the time trend. Total volatile fatty acid concentrations were higher at 19, 38, and 55% than at 0% pulp. They were higher at 38 and 55 than at 19%.

Ammonia↗

[Studies on the chemical compound of volatile oils from various species of Citrus grandis].

The volatile oils in Citrus grandis from various species were compared by GC-MS. The results showed that the content of volatile oil from Citrus grandis and Citrus grandis (L.) Osbek var. tomentosa Hort. was from 0.7% to 0.9%. Over 30 chemical compounds were identified from the volatile oils and the types of the compounds were almost the same. But gamma-terpinen could not be identified from Citrus grandis which was rich in Citrus gransis (L.) Osbeck var. tomentosa Hort.

Citrus↗

Induction of apoptosis by Citrus paradisi essential oil in human leukemic (HL-60) cells.

Limonene is a primary component of citrus essential oils (EOs) and has been reported to induce apoptosis on tumor cells. Little is known about induction of apoptosis by citrus EOs. In this study, we examined induction of apoptosis by Citrus aurantium var. dulcis (sweet orange) EO, Citrus paradisi (grapefruit) EO and Citrus limon (lemon) EO. These EOs induced apoptosis in HL-60 cells and the apoptosis activities were related to the limonene content of the EOs. Moreover, sweet orange EO and grapefruit EO may contain components besides limonene that have apoptotic activity. To identify the components with apoptotic activity, grapefruit EO was fractionated using silica gel columns, and the components were analyzed by GC-MS. The n-hexane fraction contained limonene, and the dichloromethane fraction (DF) contained aldehyde compounds and nootkatone. Decanal, octanal and citral in the DF showed strong apoptotic activity, suggesting that the aldehyde compounds induced apoptosis strongly in HL-60 cells.

Anticarcinogenic Agents↗

Efficacy assessment of Candida oleophila (strain O) and Pichia anomala (strain K) against major postharvest diseases of citrus fruits in Morocco.

Two yeasts, Candida oleophila (strain O) and Pichia anomala (strain K), were previously selected for their antagonistic activity against postharvest diseases on apples and pears. The objective of the study was to determine the efficacy of both antagonistic yeast's against wound postharvest pathogens of citrus fruits. The efficacy of both strains (applied at 10(5), 10(6) and 10(8) CFU/ml) was assessed against Penicillium digitatum and P. italicum inoculated after one hour (at a concentration of 10(5), 10(6) and 10(7) spores/ml) on citrus varieties 'clementine' and 'valencia-late'. Fruits were incubated for one week at 24 degrees C before measurement of lesion diameter. The protective levels were positively correlated with high concentration of antagonist and low concentration of pathogen. Highest protective levels (from 73 to 100%) were detected with the application of strain O or strain K at 10(8) CFU/ml whatever the pathogen (applied at 10(5) spores/ml) and the citrus variety. The antagonistic activity of both strains was also dependent on the incubation period before pathogen Inoculation. The protective level increased with time between application of the antagonist and inoculation of fungal spores. Whatever the yeast strain (10(8) CFU/ml). the protective level exceed 70% when wounded oranges were inoculated with P. digitatum or P. italicum (both at 10(6) spores/ml) 12 hours after yeast treatment. These protective levels reached 100% when the incubation period separating the antagonist application and the pathogenic inoculation was 24 hours. On the other hand, high protective levels (< 80%) were also observed against the sour rot decay on citrus variety 'clementine' caused by Geotrichum candidum inoculated at concentration of 10(6) spores/ml when strain O or strain K were applied at 10(8) CFU/ml 24 hours before pathogen. All these results support the potential practical application of both strains against major postharvest pathogens on citrus.

Candida↗

[Study on heavy metals in growing area soils and young fruit of Citrus grandis 'Tomentosa'].

OBJECTIVE: To determine the status of heavy metals contamination in growing area soils and young fruits of Citrus grandis. METHOD: Application of Nemerow's index for heavy metals assessment of soil and young fruit of Citrus grandis, as well as according to national environmental quality standard for soils and green trade standards of importing & exporting medicinal plants & preparations. RESULT: The heavy metals content of most of the growing area soils of Citrus grandis rates as first, and of the rest ones rates as second. The heavy metals content in young fruits of Citrus grandis is within the limitation of quantity of criteria. CONCLUSION: The environmental quality of growing area soils of Citrus grandis in Huazhou City is totally in good.

Arsenic↗

Enhancement of cold tolerance and inhibition of lipid peroxidation by citrus dehydrin in transgenic tobacco.

Citrus ( Citrus unshiu Marcov.) dehydrin in response to chilling stress was overexpressed in tobacco ( Nicotiana tabacum L.), and the cold stress tolerance of transgenics at low temperature was analyzed. The freezing at -4 degrees C for 3 h of 24 independent lines indicated that a phenotype expressing citrus dehydrin showed less electrolyte leakage than the control. Dehydrin protein content was correlated with freezing tolerance in transgenics. Dehydrin-expressing tobacco exhibited earlier germination and better seedling growth than the control at 15 degrees C. Cell fractionation experiments suggested that the protein was predominantly expressed in mitochondria and the soluble fraction. Malondialdehyde production enhanced by chilling stress was lower in tobacco plants expressing citrus dehydrin than in control phenotypes. Dehydrin protein, purified from Escherichia coli expressing citrus dehydrin cDNA, prevented peroxidation of soybean ( Glycine max L.) liposomes in vitro. The inhibitory activity of dehydrin against liposome oxidation was stronger than that of albumin, glutathione, proline, glycine betaine, and sucrose. These results suggest that dehydrin facilitates plant cold acclimation by acting as a radical-scavenging protein to protect membrane systems under cold stress.

Adaptation, Physiological↗

Flavanone Glycoside Biosynthesis in Citrus: Chalcone Synthase, UDP-Glucose:Flavanone-7-O-Glucosyl-Transferase and -Rhamnosyl-Transferase Activities in Cell-Free Extracts.

Previous indirect evidence suggested that the biosynthesis of flavonoids in Citrus may not proceed via the usual chalcone synthase reaction and that glycosylation occurs during chalcone formation and not afterward, as has been reported in other species. We detected chalcone-synthase and UDP-glucose:flavanone-7-O-glucosyl-transferase activities in cell-free extracts of Citrus. The glucosylated flavanone was further rhamnosylated when exogenous UDP-glucose and NADPH were added to the extract. Chalcone-synthase activity was detected in cell-free extracts derived from young leaves and fruits. Young fruits (2 millimeter diameter) had the highest chalcone synthase activity. UDP-glucose:flavanone-7-O-glucosyl-transferase activity was measured in cell-free extracts derived from young leaves and fruits of Citrus mitis and Citrus maxima. The highest UDP-glucose:flavanone-7-O-glucosyl-transferase activity was found in young C. maxima leaves. These data indicate that Citrus contains a flavonoid pathway similar to that studied in other species.

Journal Article↗

Effect of dietary wheat bran and dehydrated citrus fiber on azoxymethane-induced intestinal carcinogenesis in Fischer 344 rats.

The effect of dietary wheat bran and dehydrated citrus fiber on carcinogenesis of the colon and small intestine was studied in male F344 rats. Weanling rats were fed semi-purified diets containing 5% fat and 15% wheat bran or citrus fiber. At 7 weeks of age, all animals, except vehicle-treated controls, received weekly sc injections of 8 mg azoxymethane (AOM)/kg body weight for 10 weeks. The AOM- or vehicle-treated groups were autopsied 20 weeks after the last injection of AOM. The animals fed the wheat bran or citrus fiber diet and treated with AOM had a lower incidence (number of animals with tumors) and multiplicity (number of tumors/tumor-bearing animal) of colon tumors and tumors of the small intestine than did those fed the control diet and treated with AOM. Animals fed the diet containing wheat bran developed fewer adenomas and adenocarcinomas of the colon than did the rats fed the control diet; the number of adenomas but not the number of adenocarcinomas was reduced in rats fed the citrus pulp diet. This study thus indicates that diets containing wheat bran and citrus fiber reduced the risk of intestinal cancer.

Adenocarcinoma↗

Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination.

The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their public acceptance and future commercialization. In citrus, selection using the selectable marker gene nptII, that confers resistance to the antibiotic kanamycin, is in general very effective. An attractive alternative is offered by the MAT system (Multi-Auto-Transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with a MAT vector has been attempted in two citrus genotypes, Pineapple sweet orange (Citrus sinensis L. Osb.) and Carrizo citrange (C. sinensis L. Osb. x Poncirus trifoliata L. Raf.). Results indicated that the IPT phenotype was clearly distinguishable in sweet orange but not in citrange, and that excision was not always efficient and precise. Nevertheless, the easy visual detection of the IPT phenotype combined with the higher transformation efficiency achieved in sweet orange using this system open interesting perspectives for the generation of marker-free transgenic citrus plants.

Alkyl and Aryl Transferases↗

Evidence for host plant preference by Iphiseiodes quadripilis (Acari: Phytoseiidae) on Citrus.

In this study, we present field and laboratory evidence on the preference of Iphiseiodes quadripilis (Banks) for grapefruit (Citrus paradisi Macfadyen) leaves compared with sweet orange (Citrus sinensis (L.) Osbeck) leaves. This preference was confirmed in four orchards whether leaf samples were taken from either border trees of contiguous grapefruit or sweet orange or interior row trees with both citrus species in adjacent rows. Iphiseiodes quadripilis was most abundant in grapefruit trees in spite of the greater abundance of the Texas citrus mite, Eutetranychus banksi (McGregor) (Acari: Tetranychidae) in sweet orange trees. Similar preference responses were observed in laboratory tests using a Y-tube olfactometer whether I. quadripilis were collected from sweet orange or grapefruit. Iphiseiodes quadripilis collected from grapefruit trees showed significant preference for grapefruit over sweet orange leaves in contact choice tests using an arena of alternating leaf strips (12 mm long x 2 mm wide) of sweet orange and grapefruit. However, I. quadripilis collected from sweet orange trees did not show preference for either grapefruit or sweet orange leaves. Based on these results, grapefruit leaves foster some unknown factor or factors that retain I. quadripilis in greater numbers compared with sweet orange leaves.

Animals↗

Enhancement of beta-carotene synthesis by citrus products.

beta-Ionone, a stimulatory compound in the microbiological production of beta-carotene by mated cultures of Blakeslea trispora, could be replaced with low-cost agricultural by-products (citrus oils, citrus pulp, or citrus molasses) with as good or better carotene yields. Peak yields (81 to 129 mg of carotene per g of dry solids) were achieved in 5 days. The various citrus products tested did not change the pigments produced; all trans-beta-carotene remained the pre-dominant pigment. The acid-hydrolyzed soybean meal and corn used in previous production media could be replaced with unhydrolyzed cottonseed embryo meal and corn in a medium that also contained a natural lipid, deodorized kerosene, nonionic detergent, and a precursor.

Carotenoids↗