Coniferin—an astringent glycoside in citrus

From the peel of C. sinensis cv. Valencia, C. paradisi cv. Duncan and C. paradisi x C. reticulata cv. Murcott, coniferin, an astringent glycoside, was identified. This compound was found to be present inconcentrated Valencia juice, Murcott concentrate and orange pulp wash concentrates.     

Accumulation of Free Proline in Citrus Leaves during Cold Hardening of Young Trees in Controlled Temperature Regimes

Free proline increased in leaves of orange (Citrus sinensis [L.] Osb. cv. Valencia) and grapefruit (Citrus paradisi Macfad. cv. Star Ruby) trees on a wide range of citrus rootstocks during cold hardening. Increases in sugars accompanied proline accumulation. During cold hardening, the rate of proline accumulation was greater in old than in young leaves. In leaves of grapefruit trees kept in the dark during cold hardening, neither proline nor sugars increased and the degree of cold hardiness was less than in trees exposed to light. Like sugar accumulations, proline accumulation does not reflect specific degrees of cold hardiness in citrus cultivars.     

Citrus tissue culture: stimulation of fruit explant cultures with orange juice

In vitro growth of explant (juice vesicle or albedo tissues) cultures from citron (Citrus medica), lemon (C. limon), grapefruit (C. paradisi), sweet orange (C. sinensis), and mandarin (C. reticulata) fruits was stimulated by addition of orange juice (10% v/v optimum) to a basal medium containing Murashige and Skoog salts, 50 grams per liter sucrose, 100 milligrams per liter myo-inositol, 5 milligrams per liter thiamine·HCl, 2 milligrams per liter 2,4-dichlorophenoxyacetic acid and 0.5 milligrams per liter kinetin. In analyzing this effect of orange juice on citron explant cultures, we failed to obtain increased yields by addition of appropriate concentrations of citric acid to the basal medium but obtained growth stimulation when the medium was supplemented with juice from an “acidless” orange variety (cv. Lima). These facts suggest that some component(s) other than citric acid is involved. Addition of the inorganic ash corresponding to 10% (v/v) orange juice to the basal medium had no effect on yields. Similarly, the stimulatory effect of orange juice could not be explained based on its content of sucrose or of organic growth factors already present in the basal medium.     

Comparison of Five Populations of Tylenchulus semipenetrans to Citrus,Poncirus, and their Hybrids

The infectivity of five populations of Tylenchulus semipenetrans were compared and differentiated on 10 hosts (5 Citrus spp., 1 Poncirus trifoliata, and 4 hybrids of Citrus spp. X P. trifoliata). Differences in levels of infection and development (P = 0.01) occurred between Citrus spp. and P. trifoliata cv. ''Pomeroy'' and their three hybrids, C. paradisi X P. trifoliata cv. ''Swingle'' citruntelo and C. sinensis, cv. ''Ruby'' orange X P. trifoliata cv. ''Webber Fawcett 14-7'', and ''15-7''. Poncirus trifoliata cv. Pomeroy was susceptible to a California biotype 3 and highly resistant to the other citrus nematode populations. Low infection levels with California biotype 1, Arizona, and Florida populations on Swingle citrumelo, and the two Ruby orange hybrids indicated inherited resistance. Reproduction of the nematode population from Texas was greatest on the three hybrids, Swingle citrumelo, Ruby orange 14-7, and 15-7, from the California 1, Arizona, and Florida populations, but its comparable densities on P. trifoliata and Citrus spp. were not sufficiently different from these populations to consider it a separate biotype. California biotype 3 was sufficiently different from all other populations to be considered a different biotype, and it was named the "Poncirus biotype."     

Intra-farm diversity and evidence of genetic recombination of Citrus tristeza virus in Delhi region of India

Infection of Citrus tristeza virus (CTV) in different citrus orchards of New Delhi was detected by direct antigen coated-ELISA and RT-PCR. Sweet orange (Citrus sinensis) orchards were found to be susceptible to CTV with estimated disease incidence up to 39%. Kagzi kalan (C. lemon), Pumello (C. paradisi) and Kinnow mandarin (C. reticulata) orchards did not show CTV infection. Three CTV isolates, D1, D7 and D15 randomly selected from infected sweet orange orchards were considered for biological and molecular characterization. In the host range study, all the Delhi isolates infected Darjeeling mandarin (C. reticulata), Kagzi lime (C. aurantifolia), sour orange (C. aurantium) and sweet orange but not Kinnow mandarin. A fragment of 5??ORF1a and complete coat protein (CP) gene of these three isolates were cloned, sequenced and compared with other Indian and international CTV isolates. Delhi isolates shared 85?C92% sequence identity for 5??ORF1a fragment and 89?C91% for CP gene among them. Phylogenetic analysis segregated three Delhi isolates into three genogroups for each of 5??ORF1a fragment and CP gene, however phylogenetic relationships for both the genomic regions was incongruent. Recombination detecting program RDP3 detected CTV isolate D7 as recombinant, indicating genetic variability in CTV isolates might be the outcome of recombination events between divergent CTV sequences. An attempt was made in present study to characterize CTV isolates biologically and at genetic level, and to determine genetic diversity at farm level and study the recombination of CTV isolates in Delhi region.     

Resistance of Citrus and Related Genera to Diaphorina citri Kuwayama (Hemiptera: Liviidae)

The present study was developed to evaluate the resistance of the following genotypes of Citrus and related genera to this pest: ‘Pera,’ ‘Natal’, and ‘Washington Navel’ oranges (Citrus sinensis), ‘Marsh Seedless’ grapefruit (Citrus paradisi), hardy orange ‘Rubidoux’ (Poncirus trifoliata), kumquat (Fortunella margarita Swingle), citrumelo ‘Swingle’ (C. paradisi x P. trifoliata), and citrange ‘Troyer’ (P. trifoliata x C. sinensis). The experiments were performed in greenhouses with plants grafted onto ‘Rangpur’ lime (Citrus limonia) and placed individually in voile cages. The preference for oviposition in a no-choice test, and the effect of genotype were evaluated. The egg-adult cycle was monitored to determine the effect of genotype on the biology of the insect. Poncirus ‘Rubidoux’ was the least preferred genotype for oviposition; reduced number of eggs was also found to occur on citrange ‘Troyer’, and ‘Marsh Seedless’ was the genotype with the most eggs. No significant variation in the duration of the embryonic period was observed; however, a difference in the viability of eggs was found, with the lowest egg viabilities on ‘Swingle.’ Kumquat and ‘Marsh Seedless’ genotypes were correlated with increased durations of the nymphal phase, however, there was no difference in the survival of this phase. Fecundity of females on ‘Troyer’, ‘Swingle’, and kumquat was reduced. Considering all of the evaluated parameters, it was concluded that cultivars of sweet orange are the most susceptible genotypes to Diaphorina citri. Regarding oviposition, P. trifoliata ‘Rubidoux’ showed resistance of the antixenosis type.     

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model ??diploid embryogenic callus protoplast?+?diploid mesophyll-derived protoplast??. Protoplasts were isolated from embryogenic calli of ??Pera?? and ??Westin?? sweet orange cultivars (Citrus sinensis) and from young leaves of ??Fremont??, Nules??, and ??Thomas?? mandarins (C. reticulata), and ??Nova?? tangelo [C. reticulata?×?(C. paradisi?×?C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when ??Pera?? sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of ??Pera?? sweet orange?+???Fremont?? mandarin, 3 ??Pera?? sweet orange?+???Nules?? mandarin, and 2 ??Pera?? sweet orange?+???Nova?? tangelo, and all the diploid regenerated plants showed the ??Pera?? sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96?h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.     

Mutational analysis of substrate specificity in a <Emphasis Type="Italic">Citrus paradisi</Emphasis> flavonol 3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

Citrus paradisi 3-O-glucosyltransferase (Cp3GT, Genbank Protein ID: ACS15351) and Citrus sinensis 3-O-glucosyltransferase (Cs3GT, Genbank Protein ID: AAS00612.2) share 95% amino acid sequence identity. Cp3GT was previously established as a flavonol-specific 3-O-glucosyltransferase by direct enzymatic analysis. Cs3GT is annotated as a flavonoid-3-O-glucosyltransferase and predicted to use anthocyanidins as substrates based on gene expression analysis correlated with the accumulation of anthocyanins in C. sinensis cv. Tarocco, a blood orange variety. Mutant enzymes in which amino acids found in Cs3GT were substituted for position equivalent residues in Cp3GT were generated, heterologously expressed in yeast, and characterized for substrate specificity. Structure–function relationships were investigated for wild type and mutant glucosyltransferases by homology modelling using a crystallized Vitis vinifera anthocyanidin/flavonol 3-O-GT (PDB: 2C9Z) as template and subsequent substrate docking. All enzymes showed similar patterns for optimal temperature, pH, and UDP/metal ion inhibition with differences observed in kinetic parameters. Although changes in the activity of the mutant proteins as compared to wild type were observed, cyanidin was never efficiently accepted as a substrate.     

Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion

Somatic hybridization offers the possibility of manipulating chloroplast and mitochondrial genomes and evaluating their role on cultivar qualities in citrus. Numerous associations between Willow-leaf mandarin (Citrus deliciosa Ten.), as embryogenic parent, and sweet orange cv. Valencia (Citrus sinensis (L.) Osb.), as mesophyll parent, and between Willow-leaf mandarin (embryogenic parent) and grapefruit cv. Duncan (Citrus paradisi Macf.) (mesophyll parent) were obtained by the fusion of protoplasts induced by polyethylene glycol. Regenerated plants were characterized by flow cytometry and nuclear and mitochondrial DNA restriction fragment length polymorphism (RFLP). All plants were diploid. Diploid plants with the nuclear RFLP patterns of mandarin or sweet orange were identified in the progeny between these two parents, while only grapefruit nuclear types were found in the mandarin + grapefruit progeny. The diploid plants with the nuclear profile of the mesophyll parent originated systematically from cells formed through spontaneous association of the nuclear genome of the mesophyll parent and the mitochondrial genome of the embryogenic parent. These plants are assumed to be alloplasmic hybrids or cybrids. They were viable and have been propagated for field testing.     

Symptomatic expression of tristeza-infected citrus plants in Pakistan

Variable symptoms were recorded during a survey in the citrus trees infected or suspected to be infected with citrus tristeza virus based on ELISA tests. Sweet orange manifested main symptoms like bud union crease, pin holing, bark cracking, incompatibility, yellowing and dropping of leaves. Pin holing was particularly prevalent in Sweet orange (Citrus sinensis Osbeck), Kinnow (Citrus reticulata Blanco) and Grapefruit (Citrus paradisii Mcaf.), while incompatibility was only in Sweet orange and Grapefruit.     

Six new flavonoids from Citrus

Valencia orange (C. sinensis) and Robinson tangerine [(C. paradisi × C. reticulata) × (C. reticulata)] were examined for flavonoids. Thirteen flavonoids were isolated, six of which are new constituents of citrus peel. These are: 3,5,6,7,3′,4′-hexamethoxyflavone, 3,5,7,8,3′,4′-hexamethoxyflavone, 5-hydroxy-3,7,8,3′,4′-pentamethoxyflavone, 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone, 5,7,8,4′-tetramethoxyflavone and 5,7,8,3′,4′-pentamethoxyflavone. The latter three flavonoids are reported for the first time as natural products. A method is described for readily obtaining small quantities of 5,7,8,4′-tetramethoxy and 5,7,8,3′,4′-pentamethoxyflavones from their 5,6,7-trimethoxy analogs.     

Use of an expressed sequence tag-based method for single nucleotide polymorphism identification and discrimination of Citrus species and cultivars

Sweet orange [Citrus sinensis (L.) Osbeck] represents the most important Citrus species, followed by clementine (C. clementina Hort. ex Tan.). Citrus species and genotypes are difficult to recognize as they have a moderate level of diversity due to nucellar selection, vegetative propagation and origin by single spontaneous mutation. Despite the large number of available sequences and the existence of a draft assembly of sweet orange and clementine, there are currently no single nucleotide polymorphism (SNP) databases for Citrus species. For this purpose, the QualitySNP software was used to discover SNPs in 19 Citrus species starting from 540,000 expressed sequence tags (ESTs) assembled in 52,000 contigs. The vast majority of ESTs, contigs and SNPs were found in C. clementina and C. sinensis: 4,400 out of 16,000 contigs (27 %) of C. clementina and 4,100 out of 17,000 contigs (24 %) of C. sinensis contained putative SNPs. A total of 3,634 sequences were associated with enzymes belonging to 121 metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, among which the secondary metabolite pathway was the most represented. A total of 163 SNPs from 52 contigs and genes of specific functional categories were validated and 81 polymorphic sites were found. Thirty-seven selected SNPs, validated by Sanger sequencing, confirmed that polymorphisms were mainly between species, while poor within-species variability was discovered. This work provides a collection of 15,879 putative SNP markers that could be exploited by the Citrus community. Furthermore, the validated SNPs associated with specific genes could be used for functional genetic studies in germplasm diversity analysis, mapping and breeding.     

Distribution of Tylenchulus semipenetrans in a Texas Grapefruit Orchard

Distribution of the citrus nematode (Tylenchulus semipenetrans) was studied over 18 months in a 6-year-old orchard of grapefruit (Citrus paradisi cv. Ruby Red) on sour orange (C. aurantium) rootstock. The 1.8-ha orchard was under chemical weed control, no tillage, and flood irrigation. Highest numbers of nematodes were found in the top 15 cm of the soil profile. The nematode population peaked in April and declined to lowest levels in August and September. Numbers of nematodes were negatively correlated (r = -0.95) with soil temperatures above 29 C. Soil populations of nematodes were not correlated with soil moisture. The distribution of the nematode in the field was highly skewed and was described by a negative binomial. In this 1.8-ha block, five soil samples of 12 cores each would provide an estimate within 20% of the true nematode population mean with 95% confidence.     

Citrus rootstock responses to herbivory by larvae of the sugarcane rootstock borer weevil (Diaprepes abbreviatus)

The responses of roots to feeding by larvae of a citrus root weevil (Diaprepes abbreviatus) were investigated in Citrus grandis (L.) Osb. x Poncirus trifoliata (2N) (L.) Raf.; C. grandis x P. trifoliata (4N); P. trifoliata x C. grandis (Flying Dragon x Nakon); C. paradisi Macf. x P. trifoliata (Swingle citrumelo); C. aurantifolia (Christm.) Swingle (Citrus macrophylla); C. reticulata Blanco (Cleopatra mandarin); C. sinensis (L.) Osb. x P. trifoliata (Carrizo citrange); C. aurantium (L.) (sour orange). Chitinase, chitosanase. β-1,3-glucanase, peroxidase and lysozyme activities were measured and significant differences were observed for some of the cultivars between infested and uninfested rootstocks. Generally, increased activities were observed for chitinases and decreased activities were observed for the other enzymes measured. Numerous significant differences in hydrolase and peroxidase activities were observed between cultivars. Immunological detection revealed that new protein bands occurred in root protein extracts for six of the eight cultivars infested with larvae when an antibody to a class I potato leaf chitinase was used. Antibodies generated against two citrus chitinases of M_r 24 000 (basic chitinase cv. Valencia (C. sinensis) callus, BCVC) and M_r 28 000 (basic chitinase/lysozyme cv. Valencia callus, BCLVC) indicated that chitinases in Carrizo were induced in infested roots when the BCVC antibody was employed. These findings justify calling these proteins pathogenesis-related proteins. The chitinase that BCLVC was prepared from exhibited high lysozyme activities, and the results of western blots showed the presence of proteins at M_r 24 000 and 27 000 which are presumed to be lysozymes. Similar tests using antibodies against β-1, 3-glucanases and peroxidases indicated a diminution of protein bands that cross-reacted with infested root protein extracts compared with what occurred in controls. All of the root extracts were tested against chitosans with various percentages of acetylation; activities were linearly dependent on the amount of chitosan acetylation; i.e. the larger the amount of acetylation, the greater the activity. Significant differences in hydrolase activities were observed between infested and uninfested roots for the rootstocks using the variously acetylated substrates. All of the root protein extracts were capable of degrading peritrophic membranes removed from larvae of D. abbreviatus. This suggests that citrus chitinases may play a role in disrupting the peritrophic membrane such that ingested substances that pose a hazard to the insect may penetrate the membrane more easily.     

Carotenoids synthesized in citrus callus of different genotypes

Eight carotenoids, such as phytoene, α-carotene, violaxanthin, etc., synthesized in citrus callus of 31 genotypes were identified and determined. Though varied with genotypes, the carotenoids composition of callus derived from a certain genotype was stable, while carotenoids contents altered between sub-cultures. Some specific carotenoids were produced in calluses of limited genotypes: β-citraurin was only synthesized in calluses of Nianju tangerine (Citrus reticulata Blanco) and Page tangelo (C. reticulata × C. paradisi); while 9-Z-violaxanthin was only detected in Nianju tangerine and Skaggs Bonanza navel orange (C. sinensis L. Osbeck). Notably, the only carotenoid detected in calluses of Natsudaidai (C. aurantium L.) and other two sweet oranges (C. sinensis L. Osbeck) was phytoene. It implied that citrus calluses could be employed to produce specific carotenoids in the future. To further elucidate the characters of callus carotenoids profile, comparisons of carotenoids profiles was made among calluses, fruit tissues and leaves of four selected citrus genotypes. Results showed that lycopene was not detected in leaves and calluses; nevertheless, both citrus fruits and calluses accumulated phytoene, whereas leaves did not except those of Cara Cara navel orange. It is postulated that citrus callus featured its carotenoids profile different from fruit tissues and leaves. In conclusion, the advantages of using citrus callus as an alternative model research system in understanding the regulation of carotenogenesis have been discussed.     

Genome Wide Characterization of Short Tandem Repeat Markers in Sweet Orange (Citrus sinensis)

Sweet orange (Citrus sinensis) is one of the major cultivated and most-consumed citrus species. With the goal of enhancing the genomic resources in citrus, we surveyed, developed and characterized microsatellite markers in the ≈347 Mb sequence assembly of the sweet orange genome. A total of 50,846 SSRs were identified with a frequency of 146.4 SSRs/Mbp. Dinucleotide repeats are the most frequent repeat class and the highest density of SSRs was found in chromosome 4. SSRs are non-randomly distributed in the genome and most of the SSRs (62.02%) are located in the intergenic regions. We found that AT-rich SSRs are more frequent than GC-rich SSRs. A total number of 21,248 SSR primers were successfully developed, which represents 89 SSR markers per Mb of the genome. A subset of 950 developed SSR primer pairs were synthesized and tested by wet lab experiments on a set of 16 citrus accessions. In total we identified 534 (56.21%) polymorphic SSR markers that will be useful in citrus improvement. The number of amplified alleles ranges from 2 to 12 with an average of 4 alleles per marker and an average PIC value of 0.75. The newly developed sweet orange primer sequences, their in silico PCR products, exact position in the genome assembly and putative function are made publicly available. We present the largest number of SSR markers ever developed for a citrus species. Almost two thirds of the markers are transferable to 16 citrus relatives and may be used for constructing a high density linkage map. In addition, they are valuable for marker-assisted selection studies, population structure analyses and comparative genomic studies of C. sinensis with other citrus related species. Altogether, these markers provide a significant contribution to the citrus research community.     

Changes in citrus leaf flavonoid concentrations resulting from blight-induced zinc-deficiency

Increased flavonoid concentrations were found to correlate with the elevated levels of leaf phenolic compounds occurring in blight-induced zinc-deficient citrus. In orange (Citrus sinensis L.) leaves, the increases occurred primarily in hesperidin and diosmin, whereas in grapefruit (C. paradisi Macf.) the largest increases occurred in naringin and rhoifolin. Zinc-deficiency occurring in the blighted citrus leaves appeared to be the important contributing factor to the increased flavonoid content. Although the leaves from trees with blight were typically smaller than leaves from unaffected trees, the increased flavonoid content was not significantly due to a concentration effect. Large differences occurred in the percent increases in concentrations of certain citrus leaf flavonoids. While large increases occurred for a number of flavanone and flavone glycosides, much smaller percent increases occurred for other minor flavone glycosides, and the polymethoxyflavone aglycones. The parallel increases occurring in the concentrations of certain flavone glycosides and their flavanone analogs provide a further indication that flavanone glycosides are precursors in the biosynthesis of flavone glycosides in citrus.     

Evaluation of the Genetic Structure of Xylella fastidiosa Populations from Different Citrus sinensis Varieties

Xylella fastidiosa was isolated from sweet orange plants (Citrus sinensis) grown in two orchards in the northwest region of the Brazilian state of São Paulo. One orchard was part of a germ plasm field plot used for studies of citrus variegated chlorosis resistance, while the other was an orchard of C. sinensis cv. Pêra clones. These two collections of strains were genotypically characterized by using random amplified polymorphic DNA (RAPD) and variable number of tandem repeat (VNTR) markers. The genetic diversity (H_T) values of X. fastidiosa were similar for both sets of strains; however, H_TRAPD values were substantially lower than H_TVNTR values. The analysis of six strains per plant allowed us to identify up to three RAPD and five VNTR multilocus haplotypes colonizing one plant. Molecular analysis of variance was used to determine the extent to which population structure explained the genetic variation observed. The genetic variation observed in the X. fastidiosa strains was not related to or dependent on the different sweet orange varieties from which they had been obtained. A significant amount of the observed genetic variation could be explained by the variation between strains from different plants within the orchards and by the variation between strains within each plant. It appears, therefore, that the existence of different sweet orange varieties does not play a role in the population structure of X. fastidiosa. The consequences of these results for the management of sweet orange breeding strategies for citrus variegate chlorosis resistance are also discussed.