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Four cyclic peptides were isolated from young unshiu (unripe fruit), orange and amanatsu peelings, and their structures were established on the basis of FAB-MS (CID method) and 2D-NMR spectroscopic data, and by chemical evidence. They were each found to consist of seven or eight amino acids.
Apscaviroid nanocitri (citrus dwarfing viroid, CDVd) is a graft-transmissible pathogen that frequently coexists with other citrus viroids and affects tree vigor. This study evaluated the occurrence of citrus viroids in small growers and commercial orchards in Chile and characterized CDVd strains obtained from urban Citrus spp. trees, with unknown propagation history following the discontinuation of the national citrus certification program. A total of 301 samples from five citrus-producing regions were analyzed, revealing that CDVd is widely distributed and commonly associated with Hostuviroid impedihumuli (hop stunt viroid, HSVd). Single infections were the most frequent (41%), followed by double (7.3%) and triple infections (3.0%), indicating that mixed infections are common. CDVd strains from urban trees were characterized using biological indexing on 'Etrog' citron Arizona 861-S-1 indicator plants combined with RT-PCR detection and phylogenetic analysis. Symptom development and pathogen detection were evaluated at 6 and 18 months post-inoculation. All graft-inoculated plants developed symptoms consistent with viroid infection, whereas control plants remained asymptomatic. CDVd was consistently detected at 18 months but not at earlier stages, suggesting delayed systemic accumulation. Although Closterovirus tristezae (citrus tristeza virus, CTV) was present in the source plants, it was not detected in indicator plants at either time point, possibly associated with the higher temperatures required for viroid symptom development during bioindexing. The frequent occurrence of mixed infections highlights the epidemiological relevance of viroid interactions and supports the need for reinstatement and strengthening of the national citrus certification program.
The application of high-throughput sequencing (HTS) has accelerated the discovery of novel viruses and the genome sequencing of poorly characterised viruses in fruit crops, revealing a greater complexity of plant viromes than previously understood. Here, we report the identification and genomic characterisation of a novel emaravirus, tentatively named "citrus emaravirus 1" (CiEV1), from Citrus limon leaves collected in the North West Province of South Africa. HTS analysis identified contigs corresponding to the four conserved emaravirus genomic segments (RNA1-RNA4), each encoding characteristic proteins: RNA-dependent RNA polymerase, glycoprotein, nucleocapsid, and movement protein. Comparison of terminal sequences and phylogenetic analysis suggest placing CiEV1 within clade D of the genus Emaravirus (family Fimoviridae). To date, no association with symptoms has been identified. This is the first report of an emaravirus detected in citrus, expanding the known host range of the genus and contributing to the growing evidence of viral diversity in citrus. Further investigation is required to determine the epidemiological significance of CiEV1 in citrus orchards.
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.
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.
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.
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.
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.
A cyanide-insensitive superoxide dismutase was purified to apparent homogeneity from lemon leaves (Citrus limonum R). The enzyme was isolated from leaf extracts by ammonium sulfate salting-out, and ion-exchange, gel filtration and hydroxylapatite column chromatography. The purified Fe-SOD had a specific activity of about 1,500 U/mg and represents approximately 1.6% of the total soluble protein in lemon leaf extracts. A molecular weight of 47,500 was determined for the enzyme. Analytical gel electro-focusing of the purified preparation revealed the presence of two isozymes with pI values of 5.13 and 4.98. Metal analysis showed the presence of 1 g-atom of iron and 0.5 g-atom of manganese per mol of enzyme. The visible and UV absorption spectra of the Citrus enzyme were similar to those reported for other iron-containing SODs from different origins. The significance of the presence of Fe-SOD in higher plants is briefly discussed.
Pharmacognostical studies on the peel of Citrus reticulata cv. zhangshuensis and Citrus reticulata var. kinokuni have been carried out in comparison with four crude drugs of Chenpi. Information on the research of resources of these two drugs is provided.
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Bitterness in citrus fruit is conferred by flavanone-neohesperidosides, whose accumulation is catalyzed by a single enzyme flavanone-7-O-glucosides-1,2-rhamnosyltransferase (1,2RhaT), expressed in both leaves and fruit. To eliminate citrus bitterness, we used CRISPR/Cas9 genome editing to inactivate the 1,2RhaT gene in grapefruit (Citrus paradisi) and "Carrizo" citrange (Citrus sinensis × Citrus trifoliata). Edited lines displayed frameshift mutations that introduced premature stop codons, effectively abolishing the synthesis of the bitter neohesperidosides naringin, neohesperidin, and poncirin. Metabolomic analyses in leaves from 1,2RhaT-mutant lines confirmed the absence of bitter flavanone-neohesperidosides and a compensatory increase in the tasteless flavanone-rutinosides hesperidin, didymin, and narirutin. Since 1,2RhaT is encoded by a single gene, our findings in leaves are expected to be identical for fruit and thus demonstrate a strategy for developing non-bitter citrus cultivars while retaining health-benefitting flavonoid levels. Furthermore, cold-hardy citrus species that are currently unacceptably bitter due to high flavanone-neohesperidoside levels may become useful sources for introduction of cold-hardiness following inactivation of the 1,2RhaT gene. This approach thus paves the way for expanding grapefruit markets and breeding cold-hardy, palatable citrus varieties that are better suited to a wider range of climates.
Recent advances in the field of genome editing offer a promising avenue for targeted trait improvements in fruit trees. However, the predominant method taken for genome editing in citrus (and other fruit trees) involves the time-consuming tissue culture approach, thereby prolonging the overall citrus breeding process and subjecting it to the drawbacks associated with somaclonal variation. In this study, we introduce an in planta approach for genome editing in soil-grown citrus plants via direct transformation of young seedlings. Our editing system, abbreviated here as IPGEC (in planta genome editing in citrus), is designed to transiently co-express three key gene groups in citrus tissue via Agrobacterium tumefaciens: (i) a genome-editing catalytic group, (ii) a shoot induction and regeneration group, and (iii) a T-DNA enhanced delivery group. This integrated system significantly improves de novo shoot induction and regeneration efficiency of edited tissue. By incorporating single-guides RNA's (sgRNA's) targeting the carotenoid biosynthetic gene PHYTOENE DESATURASE (CsPDS), the IPGEC system effectively produced mutated albino shoots, confirming its ability to generate homozygous/biallelic genome-edited plants. By using high throughput screening, we provide evidence that transgene-free genome-edited plants could be obtained following the IPGEC approach. Our findings further suggest that the efficiency of specific developmental regulators in inducing transformation and regeneration rates may be cultivar-specific and therefore needs to be optimized per cultivar. Finally, targeted breeding for specific trait improvements in already successful cultivars is likely to revolutionize fruit tree breeding and will pave the way for accelerating the development of high-quality citrus cultivars.
This study was performed to determine whether synthetic ascorbic acid (AA) alone or in a natural citrus extract containing bioflavonoids, proteins, and carbohydrates was more bioavailable to human subjects. The effect of a single 500-mg ascorbate dose of the two forms and a placebo citrus extract on plasma ascorbate was examined in eight fasting subjects. A comparison of the areas under the plasma concentration-time curves showed that the citrus extract was 35% more absorbed than AA (p less than 0.001) and was more slowly absorbed than AA (p less than 0.001). In six ascorbate-saturated male subjects the ascorbate in the citrus extract produced a greater ascorbate excretion than AA alone in 24-h post-dose urine (p less than 0.05). Citrus extract ascorbate was less excreted than AA (p less than 0.05) in 12 nonsaturated subjects. Ascorbate in the citrus extract was found to be more bioavailable than AA alone in human subjects.