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PubMed2000

Citrus Blight and Other Diseases of Recalcitrant Etiology.

Several economically important diseases of unknown or recently determined cause are reviewed. Citrus blight (CB), first described over 100 years ago, was shown in 1984 to be transmitted by root-graft inoculations; the cause remains unknown and is controversial. Based on graft transmission, it is considered to be an infectious agent by some; others suggest that the cause of CB is abiotic. Citrus variegated chlorosis, although probably long present in Argentina, where it was considered to be a variant of CB, was identified as a specific disease and shown to be caused by a strain of Xylella fastidiosa after if reached epidemic levels in Brazil in 1987. Citrus psorosis, described in 1933 as the first virus disease of citrus, is perhaps one of the last to be characterized. In 1988, it was shown to be caused by a very unusual virus. The cause of lettuce big vein appears to be a viruslike agent that is transmitted by a soilborne fungus. Double-stranded RNAs were associated with the disease, suggesting it may be caused by an unidentified RNA virus. Rio Grande gummosis, dry rot root, peach tree short life, and some replant diseases may be diseases of complex etiology. Various microorganisms have been isolated from trees with these diseases, but the diseases may be attributable in part to environmental factors. Determination of the cause of these diseases of complex etiology has proven difficult, in part, because they affect only mature trees.

citrus psorosis↗
PubMed2000

Suppressive effects of citrus fruits on free radical generation and nobiletin, an anti-inflammatory polymethoxyflavonoid.

Citrus fruit intake is known to be associated with a reduction of cancer incidence. Free radicals, including superoxide (O2-) and nitric oxide (NO), are involved in some epithelial carcinogenesis processes. In the present study, we screened thirty-one citrus fruits for their suppressive activities toward three lines of free radical generating systems: 1) O2- generation by the xanthine (XA)-xanthine oxidase (XOD) system; 2) O2- generation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated human promyelocytic HL-60 cells; and 3) NO generation in murine macrophage RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon (IFN)-gamma. As a result, the inhibitory activities of peel parts were largely found to be higher than those of the corresponding juice sac parts. In particular, the peel portion of Dancy tangerine (Citrus tangerinia) showed marked anti-oxidative activities in these systems. In addition, nobiletin, a polymethoxyflavonoid isolated from C. nobilis, showed a higher anti-inflammatory activity than indomethacin in a TPA-induced edema formation test in mouse ears. These results indicate that citrus fruits could be notable sources of anti-oxidative, anti-inflammatory, and cancer preventive compounds.

Animals↗
PubMed2000

The p20 gene product of Citrus tristeza virus accumulates in the amorphous inclusion bodies.

Citrus tristeza virus (CTV) has 10 3' open reading frames (ORFs) of unknown function except for the two coat proteins. The highest produced subgenomic RNAs are those of the major coat protein gene (p25) and the 3' most genes, p20 and p23. The proteins from three ORFs, p25, p27, and p20, were examined in the yeast two-hybrid assay for the interactions between themselves and to one another. The p20 protein exhibited a high affinity for itself, suggesting that it might aggregate in infected cells. The cytopathology of CTV infections includes characteristic paracrystalline and amorphous inclusions in the phloem elements of infected citrus. Polyclonal antiserum raised against the bacterial expressed p20 gene product detected a protein of approximately 22-23 kDa, which accumulated to relatively high levels in CTV-infected citrus, but not in healthy citrus. Immunogold localization using antibodies to p20 protein showed strong and specific labeling of the amorphous inclusion bodies present in CTV-infected cells. Mesophyll protoplasts of Nicotiana benthamiana transfected with a CTV mutant containing the green fluorescent protein (GFP) ORF fused in-frame to the 3' end of p20 protein ORF expressed high levels of GFP. The fusion protein was concentrated in one specific area in the cytoplasm and lacked an organized shape. Accumulation of high levels of p20 protein in infected tissue, specific localization of the p20-GFP fusion protein, immunolocalization of p20 protein into amorphous inclusions, and strong homologous p20 protein-p20 protein interactions in the yeast-two-hybrid assay suggest that the p20 protein of CTV is a major component of the amorphous inclusion bodies present in CTV-infected cells.

Blotting, Western↗
PubMed1995

A non-photosynthetic ferredoxin gene is induced by ethylene in Citrus organs.

The sequence and expression of mRNA homologous to a cDNA encoding a non-photosynthetic ferredoxin (Fd1) from Citrus fruit was investigated. The non-photosynthetic nature of this ferredoxin was deduced from: (1) amino acid sequence alignments showing better scores with non-photosynthetic than with photosynthetic ferredoxins, (2) higher expression in tissues containing plastids other than chloroplast such as petals, young fruits, roots and peel of fully coloured fruits, and (3) the absence of light-dark regulation characteristic of photosynthetic ferredoxins. In a phylogenetic tree constructed with higher-plant ferredoxins, Citrus fruit ferredoxin clustered together with root ferredoxins and separated from the photosynthetic ferredoxins. Non photosynthetic (root and fruit) ferredoxins, but not the photosynthetic ferredoxins, have their closest homologs in cyanobacteria. Analysis of ferredoxin genomic organization suggested that non-photosynthetic ferredoxins exist in Citrus as a small gene family. Expression of Fd1 is developmentally regulated during flower opening and fruit maturation, both processes may be mediated by ethylene in Citrus. Exogenous ethylene application also induced the expression of Fd1 both in flavedo and leaves. The induction on non-photosynthetic ferredoxins could be related with the demand for reducing power in non-green, but biosynthetically active, tissues.

Amino Acid Sequence↗
PubMed1996

Molecular characterization of the mitochondrial citrate synthase gene of an acidless pummelo (Citrus maxima).

Pummelo (Citrus maxima [Burm.] Merrill) cDNAs encoding mitochondrial citrate synthase (mCS) were cloned by reverse transcription of juice-sac poly(A)+ mRNA, followed by Taq Polymerase-mediated amplification. The nucleotide sequence of the citrus gene (cit1) is 77% conserved relative to plant mRNAs for mCS. The encoded polypeptide includes a mitochondrial targeting signal at its amino terminus; all 20 putative active-site residues of the citrus enzyme are conserved. Southern hybridization showed that citrus cit1 is a single-copy gene. A polymorphism associated with cit1 did not cosegregate with fruit acidity indicating that acitric, the gene causing the acidless phenotype of pummelo 2240, is not an allele of cit1 locus. Quantitative detection of cit1 mRNA showed that transcript levels are not developmentally regulated in juice sacs; no differences were observed between high- and low-acid genotypes.

Acids↗
PubMed1993

Molecular characterization of salt-stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases.

A gene encoding for a citrus salt-stress-associated protein (Cit-SAP) was cloned from Citrus sinensis salt-treated cell suspension. The gene, designated csa, was isolated from a cDNA expression library. The partial amino acid sequence of the protein, as well as that deduced from the nucleotide sequence of csa, revealed a considerable homology to mammalian glutathione peroxidase (GP), and to clone 6P229 from tobacco protoplasts. The increased expression of Cit-SAP in NaCl-treated cultured citrus cells and in citrus plants irrigated with saline water, and its similarity to GP, raise the possibility that one of the effects of salt stress in plants may be the increase of the level of free radicals.

Amino Acid Sequence↗
PubMed2006

An effective and low-cost culture medium for isolation and growth of Xylella fastidiosa from citrus and coffee plants.

Buffered charcoal-yeast extract medium (BCYE) has been used for isolation of Xylella fastidiosa from citrus (Citrus sinensis) and coffee (Coffea arabica) plants affected by citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). BCYE is composed of ACES (2-[2-amino-2oxoethyl) amino]-ethanesulfonic acid) buffer, activated charcoal, yeast extract, L-cysteine, ferric pyrophosphate, and agar. ACES buffer is costly and not always commercially available in Brazil, and the L-cysteine and ferric pyrophosphate need to be filter sterilized in 0.22-mum pore membranes before inclusion in the medium. Omission of L-cysteine, addition of magnesium sulfate, and replacements of ACES and ferric pyrophosphate for potassium phosphate and ferrous sulfate resulted in an effective, less expensive, and entirely autoclavable medium, named phosphate buffered charcoal-yeast extract medium (PCYE). The final cost of PCYE was approximately one tenth that of BCYE. Its effectiveness was tested for the isolation of X. fastidiosa from symptomatic leaves collected from 52 citrus plants affected by CVC and 43 coffee plants affected by CLS. PCYE was as effective as BCYE and has been used routinely in our and other laboratories for isolation, growth, and quantification of X. fastidiosa from plant tissues.

Brazil↗
PubMed2004

Targeted cybridization in citrus: transfer of Satsuma cytoplasm to seedy cultivars for potential seedlessness.

CMS (cytoplasmic male sterility) can be controlled by the mitochondrion genome in higher plants, including Satsuma mandarin. Somatic fusion experiments in citrus combining embryogenic callus protoplasts of one parent with leaf protoplasts of a second parent often produce cybrid plants of the leaf parent, a phenomenon occurring most often with interspecific fusion combinations. In an attempt to practically exploit this cybridization phenomenon, we conducted somatic fusion experiments combining embryogenic suspension-derived protoplasts of Satsuma mandarin, Citrus unshiu Marc. cv. Guoqing No. 1 (G1), a male-sterile cultivar, with leaf protoplasts of other seedy types--Hirado Buntan Pink pummelo (HBP) [Citrus grandis (L.) Osbeck], Sunburst mandarin (C. reticulata Blanco), Orie Lee hybrid (C. reticulata cv. Clementine x Murcott tangor), and Murcott tangor [C. reticulata x C. sinensis (L.) Osbeck], respectively--in an attempt to generate seedless cybrids by the targeted transfer of CMS. The genetic identities of regenerated plants from all four parental combinations were determined by flow cytometry, SSR, CAPS (or PCR-RFLP), RFLP, and chloroplast-SSR analyses. Regenerated plants from the first three parental combinations were diploids, and the cybrid nature of G1 + HBP with the mitochondrion genome from G1 and the chloroplast genome from HBP was confirmed, whereas the cybrid nature of the remaining two combinations was difficult to confirm because of the close phylogenetic relatedness of both fusion parents, as expected. Plants from G1 + Murcott were confirmed as tetraploid somatic hybrids. This is the first report of targeted citrus cybrid production by symmetric fusion with male-sterile Satsuma as the callus parent and other seedy cultivars as the leaf parents.

Cell Fusion↗
PubMed2006

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 x 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.

Cell Fusion↗
PubMed2025

Transgene-free genome editing in citrus and poplar trees using positive and negative selection markers.

Transgene-free genome editing of the gene of interest in citrus and poplar has been achieved by co-editing the ALS gene via transient transgene expression of an efficient cytosine base editor. CRISPR-Cas genome editing systems have been widely used in plants. However, such genome-edited plants are nearly always transgenic in the first generation when Agrobacterium-mediated transformation is used. Transgene-free genome-edited plants are valuable for genetic analysis and breeding as well as simplifying regulatory approval. It can be challenging to generate transgene-free genome-edited plants in vegetatively propagated or perennial plants. To advance transgene-free genome editing in citrus and poplar, we investigated a co-editing strategy using an efficient cytosine base editor (CBE) to edit the ALS gene to confer herbicide resistance combined with transient transgene expression and potential mobile RNA-based movement of CBE transcripts to neighboring, non-transgenic cells. An FCY-UPP based cytotoxin system was used to select non-transgenic plants that survive after culturing on 5-FC containing medium. While the editing efficiency is higher in poplar than in citrus, our results show that the CBE-based co-editing strategy works in both citrus and poplar, albeit with low efficiency for biallelic edits. Unexpectedly, the addition of the TLS mobile RNA sequence reduced genome editing efficiency in both transgenic and non-transgenic plants. Although a small fraction of escaping plants is detected in both positive and negative selection processes, our data demonstrate a promising approach for generating transgene-free base-edited plants.

Populus↗
PubMed2005

Larval Cryptothelea gloverii (Lepidoptera: Psychidae), an arthropod predator and herbivore on Florida citrus.

The orange bagworm (OBW), Cryptothelea gloverii (Packard) (Lepidoptera: Psychidae) was previously reported feeding on citrus fruit and foliage and preying upon the camphor scale Pseudaonidia duplex (Cockerell) (Homoptera: Coccidae). In this study using laboratory assays, OBW preyed upon citrus rust mite, Phyllocoptruta oleivora (Ashmead) (Acari: Eriophyidae) and consumed eggs and adults of both P. oleivora and Panonychus citri (McGregor) (Acari: Tetranychidae), two important pest mites on Florida citrus. OBW was also observed to feed on the purple scale, Lepidosaphes beckii (Newman) (Homoptera: Diaspididae) and on a fungus (Penicillium sp.). OBW fed on orange and grapefruit leaves by starting from the border and eating part of the leaf, by chewing holes, or consuming the outer epithelium of either the axial or abaxial surface of the leaf without penetrating through the leaf. OBW was observed in orange orchards in association with fruit extensively russeted by P. oleivora feeding. Laboratory assays revealed that OBW larvae preferred to feed on oranges infested with P. oleivora rather than on clean fruits that were free of mite feeding damage. Feeding damage to citrus fruit by OBW larvae results in one to several holes being eaten into the rind or albedo, without damage to the fruit sacs.

Animals↗
PubMed2006

Global analysis of gene expression during development and ripening of citrus fruit flesh. A proposed mechanism for citric Acid utilization.

Microarrays of cDNA have been used to examine expression changes of 7000 genes during development and ripening of the fruit flesh of self-incompatible Citrus clementina, a non-climateric species. The data indicated that 2243 putative unigenes showed significant expression changes. Functional classification revealed that genes encoding for regulatory proteins were significantly overrepresented in the up-regulated gene clusters. The transcriptomic study together with the analyses of selected metabolites highlighted key physiological processes occurring during citrus fruit development and ripening such as water accumulation, carbohydrate build-up, acid reduction, pigment substitutions (carotenoid accumulation and chlorophyll decreases) and ascorbic acid diminution. Often, the combined analyses strongly suggested prevalence of specific metabolic alternatives. This observation has been exemplified with the proposal for a mechanism for citrate utilization, a process of much importance in citrus industry. Microarray data validated by real-time RT-PCR suggested that citrate was sequentially metabolyzed to isocitrate, 2-oxoglutarate and glutamate. Thereafter, glutamate was both utilized for glutamine production and catabolyzed through the gamma-aminobutirate (GABA) shunt (GABA --> succinate semialdehyde --> succinate). This last observation appears to be of special relevance since it links the proton consuming reaction glutamate + H(+)--> GABA + CO(2) with high acid levels. GG-MS determinations showed that glutamate was constant while GABA levels decreased at ripening in agreement with a feasible activation of the GABA shunt during acid catabolism. This suggestion provides a convincing explanation for the strong reduction of both citrate and cytoplasmatic acidity that takes place in citrus fruit flesh during development and ripening.

Amino Acids↗
PubMed2006

Citrus flavonoids in fruit and traditional Chinese medicinal food ingredients in China.

Flavonoids-enriched tissues of citrus such as peel, immature fruit and flower are consumed as culinary seasonings, tea ingredients in China for centuries. This HPLC quantitative study on the five citrus flavonoids, naringin, hesperidin, neohesperidin, sinensetin and nobiletin on a wide range of Chinese citrus fruits and several Traditional Chinese Medicinal food ingredients in East China, revealed a great diversity in flavonoid composition. Huyou peel (C. paradisi cv. Changshanhuyou) was found to be the best naringin (3.25%) and neohesperidin (2.76%) source; C. aurantium, a major ingredient of several citrus-related TCM, is also a suitable source of naringin and neohesperidin, and a good juice source for flavanone glycosides; the peel of Wenzhoumiju (C. unshiu) is one of the richest local species in hesperidin (up to 6.25%); Zaoju (C. subcompressa) has the highest content of nobiletin (0.59%), a polymethoxylated flavone. LC-ES-MS analysis of Zanthoxylum genus for flavonoids revealed for the first time the presence of significant amounts (0.74%) of hesperidin in the root of Liangmianzhen (Z. nitidum (Roxb.) DC), a relative of Sichuan pepper, which is a spice widely used in China.

China↗
PubMedDate not supplied

Citrus psorosis is probably caused by a bipartite ssRNA virus.

Isolate 90-1-1 Concordia (Argentina) of the citrus psorosis agent was graft-transmitted to citrus and mechanically transmitted to Chenopodium quinoa, which was used as a local lesion assay host. Infected citrus and C. quinoa plant lesions were used as starting materials for the purification of the psorosis-associated agent. In extracts partially purified by differential centrifugation, infectivity was abolished by RNase treatment, even in 0.3 M NaCl, indicating that ssRNA is required for biological activity. The total loss of infectivity produced by proteinase K treatment and the decline in infectivity caused by phenol extraction indicated that protein may be essential for infectivity. When partially purified extracts were subjected to sucrose density gradient centrifugation, infectivity on C. quinoa from certain 2-fraction combinations was higher than expected, compared to the infectivity of the individual fractions. Therefore, infectivity was not associated with a single component but with the combination of at least two components which were distinguishable on sedimentation. The infectious material was present in the top and bottom zones of a sucrose gradient, which on further purification by a second gradient and agarose gel electrophoresis, revealed the presence of a 50-kDa protein. This protein was absent in comparable gradient fractions from healthy plants, and therefore most likely represented the capsid protein of both the top and bottom sucrose gradient zone components. Taken together, these results led to the conclusion that the citrus-psorosis-associated virus (CPsAV) is a multipartite virus, containing ssRNA and a 50-kDa coat protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Argentina↗
PubMed2005

Endogenous free polyamines and their role in fruit set of low and high parthenocarpic ability citrus cultivars.

Endogenous free polyamines (PAs), putrescine, spermidine and spermine, from developing fruitlets of Citrus species (Citrus unshiu Marc. and Citrus clementina Hort ex Tanaka) which differ in their parthenocarpic ability, and from uniflowered leafy and leafless inflorescences differing in their ability to set, have been determined by dansylation and separation of dansyl derivatives by HPLC. No significant differences in PAs content were observed between species or between leafy and leafless inflorescences which, nevertheless, significantly differed in fruit set. However, significant differences in their content were found in developing fruitlets, depending on the preceding flowering intensity of the tree and on the fruitlet load. These results suggest that, in Citrus, PAs may act as a nitrogen source rather than a regulator of fruit set.

Citrus↗
PubMed2006

Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing.

Citrus huanglongbing (HLB, ex greening) is one of the most serious diseases of citrus. Different forms of the disease are caused by different Candidatus Liberobacter species, Candidatus Liberibacter asiaticus (Las), Ca. L. africanus (Laf) and Ca. L. americanus (Lam). The pathogen is transmitted by psyllid insects and by budding with contaminated plant materials. The vector psyllid Diaphorina citri can transmit both Las and Lam. Establishment of this vector into Florida, reports of Lam and Las in Brazil in 2004, and recent confirmation of HLB in Florida in September 2005 is of great concern to the citrus industry. Research on HLB has been hampered by the unculturable nature of the causal bacterium in artificial media. It has also been difficult to detect and identify the pathogens, possibly because of low concentration and uneven distribution in host plants and vector psyllids. In this study, we developed quantitative TaqMan PCR using 16S rDNA-based TaqMan primer-probe sets specific to the different Ca. Liberobacter spp. An additional primer-probe set based on plant cytochrome oxidase (COX) was used as a positive internal control to assess the quality of the DNA extracts. The assays do not cross-react with other pathogens or endophytes commonly resident in citrus plants, and are very sensitive. HLB pathogen DNA was successfully amplified from the equivalent of 20 ng of midrib tissue from symptomatic leaves. The consistent results of the assays with DNA extracted from plants infected by various Ca. Liberibacter species grown in greenhouses and in the field demonstrated a degree of reproducibility for these TaqMan assays. Inhibitors of the PCR that are frequently present in plant extracts did not affect the assay results. The population of the pathogens was estimated to be 5 x 10(7) and 2 x 10(6) cells/g of fresh midribs of symptomatic sweet orange leaves infected by Las and Lam, respectively. The ratio of pathogen DNA to host plant DNA was estimated by to be 1:13,000 (w/w) and 1:1000 (c/c: target copy/target copy) in DNA extracts obtained by a standard CTAB method. Our rapid, sensitive and specific TaqMan PCR assay for the detection, identification and quantification of Ca. Liberibacter species has been successfully used in the confirmation of HLB caused by Las in Florida, and will be very useful for a broad range of research programs as well as the regulatory response and management of HLB disease.

Alphaproteobacteria↗
PubMed2000

Molecular cloning and characterization of a novel gene encoding limonoid UDP-glucosyltransferase in Citrus.

We isolated a cDNA clone encoding limonoid UDP-glucosyltransferase (limonoid GTase) from the albedo of Satsuma mandarin (Citrus unshiu Marc.) and investigated the contribution to limonoid glucoside accumulation in fruit. The isolated cDNA clone (CitLGT) was 1732 bp in length encoding 511 deduced amino acids with a predicted molecular mass of 57.5 kDa. The products of in vitro translation from an expression vector had the limonoid GTase activity. Southern blot analysis of genomic DNA indicated that CitLGT was present as a single copy gene in the Citrus genome. The amount of transcript corresponding to CitLGT mRNA changed the same way as the fluctuation of limonin glucoside content during fruit development of navel orange (Citrus sinensis Osb.). This indicates that the transcription of CitLGT regulates the conversion of limonoid aglycones to glucosides in citrus fruit.

Amino Acid Sequence↗
PubMed1989

Allergic contact dermatitis resulting from sensitivity to citrus peel, geraniol, and citral.

A bartender with hand dermatitis had allergic contact sensitivity to the skin of lemon, lime, and orange but not to their juices. Although most reported cases of citrus peel allergy are due to d-limonene, for our patient, reactions to patch tests for geraniol and citral, two minor components of citrus peel oil, were positive, whereas those for d-limonene were negative. Contact allergy to citrus peel oil should be considered in patients with hand dermatitis who are occupationally exposed to citrus fruits.

Acyclic Monoterpenes↗