Cultivated Citrus Species Research Articles

Improving fruit set and quality of Daisy mandarin through foliar application of nutrients

Daisy mandarin is a widely cultivated citrus species valued for its nutritional qualities and organoleptic properties, favored for both fresh consumption and processing. Optimal growth and fruit production is depend upon the availability of essential nutrients. However, soils of the central zones of Punjab are deficient in potassium which hinders the quality and production and their deficiency impairs physiological and biochemical processes, reducing fruit set, yield and quality. Potassium and calcium are vital for metabolic functions and cell wall integrity. A deficiency in any of these nutrients can results in smaller fruits, lower fruit retention and diminished organoleptic and nutritional qualities, such as reduced antioxidants content. So that, a research was conducted to evaluate the effect of different concentrations of potassium nitrate, potassium sulfate, calcium nitrate and boric acid along with paddy straw mulching on Daisy mandarin during the years 2021–22. The pooled data of this research showed that potassium nitrate (2%), is the best treatment with maximum fruit set (14.37%) and retention (72.35%) in Daisy mandarin. Additionally, the average fruit weight (207.50 g), number of fruits (162), fruit yield (33.79 kg/tree), fruit length (6.25 cm) and breadth (6.73 cm) also enhanced. Furthermore, potassium nitrate (2%) application increased the antioxidant activity (39.43%), phenolic content (230.00 mg kg−1 FW) and flavonoid content (3.69 mg RE g−1 FW). The findings suggest that application of potassium nitrate presents a promising strategy for enhancing fruit set, yield and quality of Daisy mandarin, providing valuable insights for improved cultivation practices.

Direct Organogenesis of Citrus Cultivars from Shoot Tip Nodal Segments.

Citrus cultivar improvement via conventional breeding strategies is impeded by factors related to its reproductive biology. The orange is a hybrid between pomelo (Citrus maxima) and mandarin (Citrus reticulata). Among various orange cultivars, Valencia oranges have a bit of bitter tang mixed in with their sweetness, as Navel oranges are, the most widely cultivated citrus species, quite sweeter, and also don't contain any seeds. Tangelo mandarin orange cultivar is a hybrid of C. reticulata × C. maxima or × C. paradisi. The present study was undertaken to optimize the hormonal composition of the media with regard to plant growth regulators for in vitro propagation of sweet orange cultivars from nodal segment explants. The nodal segment explants were collected from three citrus cultivars, Washington Navel, Valencia and Tangelo. Murashige and Skoog (MS) medium supplemented with sucrose and different concentrations of growth regulators was used for shoot proliferation and root induction, and the optimum medium composition was assessed. The patent for Citrus Tissue Culture was obtained from the Office of Research Affairs, Haramaya University. The results indicate that the highest shoot response was recorded for Washington's navel with maximum shoot proliferation rate (99.75%), shoot number per explant (1.76), shoot length (10.70 cm), leaf number per explants (3.54) after three weeks of culture. In all experiments, no growth was observed for the basal MS medium. Phytohormone combinations of IAA (1.2 mg/L) and kinetin (2.0 mg/L) were found to be the best for shoot proliferation. Among the cultivars, there were significant differences for the highest rooting rate (81.255), root number (2.22), and root length (2.95 cm) variables for Washington Navel. The lowest rooting rate (48.45%), root number (1.47) and root length (2.26 cm) were observed for Valencia. The highest rooting rate (84.90%), root number per microshoot (2.22) and root length (3.05 cm) was on MS medium supplemented with 1.5 mg/L NAA. A comparison of different concentrations of IAA and NAA on root induction of microshoots from nodal segments of citrus cultivars demonstrated NAA was a more effective hormone than IAA.

Hexyl Gallate Loaded Microgels Enable Efficient Protection Against Citrus Canker

AbstractThe development of efficient and environmentally friendly plant protection systems is one of the major challenges for a sustainable agriculture of the future. Citrus canker caused by the pathogen Xanthomonas citri subsp. citri (X. citri) affects all cultivated citrus species worldwide and is responsible for enormous economic losses and restrictions in international trade. Currently used commercial copper‐based formulations are the origin of contamination for soil and groundwater, affecting local ecosystems and human health. A copper‐free sustainable microgel‐based plant protection system able to efficiently combat X. citri is developed. Microgels decorated with anchor peptides exhibit strong non‐covalent attachment to the surface of orange leaves and have the ability to release hexyl gallate, which inhibits the growth and spreading of pathogens. The tailored design of the microgel network allows high loadings of hexyl gallate (up to 40 wt.%) and a controlled release of gallates from microgels that provide long‐term protection. The antibacterial activity of the hexyl gallate‐loaded microgels is demonstrated by various in vitro assays as well as on orange plants in greenhouse settings. The experimental results suggest that the developed sustainable microgel‐based plant protection system for citrus trees allows efficient abatement of X. citri and reduces environmental pollution caused by copper formulations.

A Decade-Long Quantitative–Qualitative Characterization of 18 Lemon Cultivars

Together with lime, lemon is recognized as the third most extensively cultivated citrus species worldwide, trailing only behind orange and mandarin. The enhancement in the economic value of lemons as a valuable resource in Southern Italy depends by the competitiveness of local lemon production in both domestic and international markets. This necessitates conducting a comprehensive study that thoroughly explores the available indigenous germplasm at the local and national levels. This study aims to identify the most promising Sicilian and Campanian cultivars that are well suited for both fresh fruit consumption and the production of processed goods. This re-study assumes even greater importance considering the ongoing climate changes, as environmental stresses significantly impact the ripening process and the timing of fruit development. Our study has highlighted a notable diversity among the 18 investigated lemon cultivars, particularly highlighting specific cultivars that possess desirable attributes for fresh consumption. The cultivars that showed the greatest cumulative production over the 10 years of the study were Erice with 467.89 kg/tree and Femminello Siracusano 2KR with 408.44 kg. Notably, cultivars like Segesta, Erice, and Kamarina have exhibited higher percentages of juice content ranging from 27.30% to 31.08%. These cultivars show great potential for abundant juice content and optimal acidity levels for direct consumption. On the other hand, cultivars characterized by enhanced yield, such as Femminello Siracusano 2KR, Femminello Fior d'Arancio m79, and Erice, may prove to be particularly well suited to produce processed goods. Overall, our findings provide valuable insights into some qualitative parameters of lemon cultivars, important either for fresh consumption or for transformed products.

Optimization of Hormonal Compositions of Media in In Vitro Propagation of Orange Cultivars from Shoot Tip Nodal Segments

Orange is one of the most horticulturally important and widely cultivated Citrus species. Citrus cultivar improvement via conventional breeding strategies is normally impeded by factors related to its reproductive biology. The present study was undertaken to investigate the optimization of growth regulators’ composition of media in in vitro propagation of orange cultivar from nodal segment explants. The nodal segment explants were collected from sweet orange cultivars. The MS medium supplemented with sucrose and different concentrations of growth regulators were used for shoot proliferation and root induction. The optimum compositions of growth regulators in MS medium were assessed. The result indicated that the highest shoot response was recorded for Washington naval orange with maximum shoot proliferation rate (99.75%), shoot number per explant (3.10), shoot length (10.70 cm), leaf number per explants (12.50) after three weeks of culture. In all experiments, no growth was observed for the basal MS medium. Phytohormones combinations of indole-3-acetic acid, IAA (1.2 mg/L), and kinetin (2.0 mg/L) were found to be the best for shoot proliferation. Among the cultivars, significantly, the highest rooting rate (81.25%), root number (4.95), and root length (2.95 cm) were recorded for Washington naval orange cultivar. The least rooting rate (48.45%), root number (3.55), and root length (2.26 cm) were observed from the Valencia cultivar. Significantly, the highest rooting rate (84.90%), root number per microshoot (5.20), and root length (3.05 cm) for MS medium supplemented with 1.5 mg/L 1-naphthalene acetic acid (NAA). The comparison of different concentrations of IAA and NAA on root induction of microshoots from nodal segments of sweet orange cultivars demonstrated NAA as the more effective hormone than IAA.

Brassinosteroids Improve Postharvest Quality, Antioxidant Compounds, and Reduce Chilling Injury in ‘Arrayana’ Mandarin Fruits under Cold Storage

‘Arrayana’ mandarin is one of the most cultivated citrus species in Colombia, but this fruit has a short postharvest life and is sensitive to chilling injury (CI) during cold storage. Generating strategies that decrease CI to mandarin can reduce quantitative and qualitative losses postharvest. Brassinosteroids (BR) have been used as a sustainable technology to alleviate CI in fruits and improve postharvest quality. This study evaluated the effect of applying the 24-epibrasinolide analogue (EBR), at doses of 5 mg L−1; DI-31 analogue, at 5 and 10 mg L−1; and control, on the main physical and biochemical characteristics of ‘Arrayana’ mandarin stored at 4 °C for 40 days and, subsequently, 7 days at room temperature (shelf life). The application of EBR and DI-31 analogues reduced the appearance of CI in the exocarp of ‘Arrayana’ mandarin fruits by reducing electrolyte leakage, maintaining membrane integrity, and increasing antioxidant activity and phenol content at the end of cold storage and shelf life. This was especially pronounced with 5 mg L−1 of EBR. Similarly, the BR maintained the postharvest quality of mandarins by reducing weight loss, respiratory intensity, and chlorophyll degradation; increasing β-carotene; and maintaining titratable acidity and soluble solids. Our research reports, for the first time, CI tolerance in Arrayana mandarin using natural (EBR) and spirostanic (DI-31) analogues and illustrates the tolerance functionality of the DI-31 analogue on CI in the fruit postharvest.

Realizing certainty in an uncertain future climate: modeling suitable areas for conserving wild Citrus species under different change scenarios in India.

Citrus is an important horticultural crop of India and is often prone to diseases, particularly under increased temperature scenarios. For developing disease-resistant Citrus varieties, conservation of wild relatives is extremely important. However, our knowledge on temperature tolerance of these wild relatives of Citrus to varied climate change scenarios is extremely limited. Therefore, we determined the climatic niche of six wild relatives of cultivated Citrus species (C. indica Tanaka, C. karna Rafin., C. latipes (Swingle) Tanaka, C. macroptera Montrouz., C. medica L., and C. sinensis (L.) Osbeck.) and identified the geographical areas in India that would remain climatically stable in future through ecological niche modeling (ENM). Raster data on 19 bioclimatic variables with a resolution of 0.04° were used to generate niche models for each Citrus species that delineated their potential distribution areas. Future species distribution predictions for the year 2050 were made using the climate change scenarios from the most appropriate climate models, i.e., IPSL-CM5A-LR and NIMR-HADGEM2-AO with four Representative Concentration Pathways (RCPs). Ensemble of current and future projections was used to identify climatically stable areas for each species. Precipitation-related bioclimatic variables were the key climatic determinants for the modeled distribution pattern. The consensus of current and future projections suggests that most areas with stable climates for the species in the future would be available in the northeastern states of Arunachal Pradesh, Meghalaya, Mizoram, and Tripura. Efforts for in situ conservation and establishment of germplasm banks and citrus orchards may be encouraged in these identified areas.

Identification of Field Tolerance and Resistance to Mal Secco Disease in a Citrus Germplasm Collection in Sicily

Mal secco is a tracheomycotic disease caused by the fungus Plenodomus tracheiphilus (Petri) Gruyter, Aveskamp, and Verkley that has caused severe damage and loss of yield in the citrus industry in the Mediterranean area, for 100 years. While the disease can affect different cultivated citrus species, lemon (C. × limon var. limon (L.) Burm. f.) and citron are the most susceptible. The identification of resistant or field-tolerant clones and hybrids is a major goal for lemon growers and breeders. To identify sources of resistance or tolerance to the disease, we performed a phenotypic survey on a lemon and lemon-like open-field germplasm planted at CREA (Research Centre for Olive, Fruit and Citrus Crops), Italy, in an area with high pathogen pressure. Phenotyping was performed visually, four times, for three consecutive years, on a total of 50 accessions, with two or three replicate trees per accession. Moreover, molecular screening based on real-time PCR was performed, for two consecutive years, on twigs, young leaves, and mature leaves of all plants, to detect the pathogen in the absence of clear symptoms. The accessions were categorized into seven groups based on the presence of visual symptoms, real-time PCR pathogen detection, and canopy volume. The results revealed sources of tolerance in lemon and citron hybrids. The molecular screening identified P. tracheiphilus in all lemon clones, with mean Ct values ranging from 17 to 39. The screening also identified P. tracheiphilus in clones without clear symptoms, indicating their ability to tolerate the disease. Moreover, a strong negative correlation was found between the Ct values in twigs and symptom severity (r = −0.72). This indicates that the DNA from twigs is the most appropriate for use in performing reliable phenotyping of mal secco susceptibility in adult plants. An autotetraploid lemon (Doppio Lentini) seems to be immune to the disease, under natural pressure, since P. tracheiphilus was not detected by real-time PCR and visual screening. Overall, the data obtained are a valuable resource for identifying both the most tolerant lemon varieties suitable for areas with high pathogen pressure and the best breeding parents for the introgression of resistance genes into lemon genotypes.

Phylogenetic Origin of Primary and Secondary Metabolic Pathway Genes Revealed by C. maxima and C. reticulata Diagnostic SNPs.

Modern cultivated Citrus species and varieties result from interspecific hybridization between four ancestral taxa. Among them, Citrus maxima and Citrus reticulata, closely associated with the pummelo and mandarin horticultural groups, respectively, were particularly important as the progenitors of sour and sweet oranges (Citrus aurantium and Citrus sinensis), grapefruits (Citrus paradisi), and hybrid types resulting from modern breeding programs (tangors, tangelos, and orangelos). The differentiation between the four ancestral taxa and the phylogenomic structure of modern varieties widely drive the phenotypic diversity’s organization. In particular, strong phenotypic differences exist in the coloration and sweetness and represent important criteria for breeders. In this context, focusing on the genes of the sugar, carotenoid, and chlorophyll biosynthesis pathways, the aim of this work was to develop a set of diagnostic single-nucleotide polymorphism (SNP) markers to distinguish the ancestral haplotypes of C. maxima and C. reticulata and to provide information at the intraspecific diversity level (within C. reticulata or C. maxima). In silico analysis allowed the identification of 3,347 SNPs from selected genes. Among them, 1,024 were detected as potential differentiation markers between C. reticulata and C. maxima. A total of 115 SNPs were successfully developed using a competitive PCR technology. Their transferability among all Citrus species and the true citrus genera was very good, with only 0.87% of missing data. The ancestral alleles of the SNPs were identified, and we validated the usefulness of the developed markers for tracing the ancestral haplotype in large germplasm collections and sexually recombined progeny issued from the C. reticulata/C. maxima admixture gene pool. These markers will pave the way for targeted association studies based on ancestral haplotypes.

Pollen Variations among some Cultivated Citrus Species and its Related Genera in Egypt

The present investigation aims to study the pollen morphology and ultrastructure of pollen grain characteristics for nine Citrus species and three related genera cultivated in Egypt. The pollen grains were photographed by using both Light Microscope (LM) and Scanning Electron Microscope (SEM). Twelve qualitative and quantitative pollen morphological characters were used to differentiate among the studied taxa. Statistical analysis of palynological data indicated that the pollen size, shape, colpi length, apertures number and type, ora size, amb shape, mesocolpium diameter, and the exine ornamentation were the most distinguished characters in the circumscription of the studied taxa and were of taxonomic value. On the contrary, the other studied pollen characters including the ratio between Polar length/Equatorial diameter (P/E), ora shape and exine thickness were not found to be of taxonomic value in the differentiation among the closely related taxa of Citrus, Fortunella margarita, Limequat hybrid and Poncirus trifoliate in the present study.

Fruit flavonoid variation between and within four cultivated Citrus species evaluated by UPLC-PDA system

Citrus fruits are rich in flavonoids and well-recognized for their health values. In order to describe the flavonoid characters of a citrus fruit type accurately, the flavonoid in fruits (peels and pulps) of four most commonly cultivated Citrus species represented by sweet orange (Citrus sinensis Osbeck cv. Newhall), lemon (C. limon Burm.f. cv. Eureka), pummelo (C. grandis Osbeck cv. Liangpingyou) and tangerine (C. tangerina Tanaka cv. Dahongpao), respectively, were studied using a new UPLC-PDA method. In this study, the results showed that different cultivated species were characterized by different dominant flavonoid compounds, such as naringin, erocitrin and hesperidin were the most predominant components of pummelo, lemon, mandarin and sweet orange, respectively. Nine polymethoxylated flavones (PMFs) were identified in the peels of sweet orange and mandarin using this new method, and nobiletin was proved to be the most dominant one. The flavonoid contents varied obviously among different fruit samples even collected from the same production area and the same cultivars. However, it is important to note that all the samples of the same cultivated species grouped in the same cluster in three dimensional scatter plots of principal components analysis (PCA). This study emphasized that the UPLC-PDA method is a simple, rapid and accurate method for citrus flavonoids analysis.

Proteomics analysis reveals novel host molecular mechanisms associated with thermotherapy of 'Ca. Liberibacter asiaticus'-infected citrus plants.

BackgroundCitrus Huanglongbing (HLB), which is linked to the bacterial pathogen ‘Ca. Liberibacter asiaticus’ (Las), is the most devastating disease of citrus plants, and longer-term control measures via breeding or genetic engineering have been unwieldy because all cultivated citrus species are susceptible to the disease. However, the degree of susceptibility varies among citrus species, which has prompted efforts to identify potential Las resistance/tolerance-related genes in citrus plants for application in breeding or genetic engineering programs. Plant exposure to one form of stress has been shown to serendipitously induce innate resistance to other forms of stress and a recent study showed that continuous heat treatment (40 to 42 °C) reduced Las titer and HLB-associated symptoms in citrus seedlings. The goal of the present study was to apply comparative proteomics analysis via 2-DE and mass spectrometry to elucidate the molecular processes associated with heat-induced mitigation of HLB in citrus plants. Healthy or Las-infected citrus grapefruit plants were exposed to room temperature or to continuous heat treatment of 40 °C for 6 days.ResultsAn exhaustive total protein extraction process facilitated the identification of 107 differentially-expressed proteins in response to Las and/or heat treatment, which included a strong up-regulation of chaperones including small (23.6, 18.5 and 17.9 kDa) heat shock proteins, a HSP70-like protein and a ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO)-binding 60 kDa chaperonin, particularly in response to heat treatment. Other proteins that were generally down-regulated due to Las infection but up-regulated in response to heat treatment include RuBisCO activase, chlorophyll a/b binding protein, glucosidase II beta subunit-like protein, a putative lipoxygenase protein, a ferritin-like protein, and a glutathione S-transferase.ConclusionsThe differentially-expressed proteins identified in this study highlights a premier characterization of the molecular mechanisms potentially involved in the reversal of Las-induced pathogenicity processes in citrus plants and are hence proposed targets for application towards the development of cisgenic Las-resistant/tolerant citrus plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0942-x) contains supplementary material, which is available to authorized users.

Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markers.

The origin of limes and lemons has been a source of conflicting taxonomic opinions. Biochemical studies, numerical taxonomy and recent molecular studies suggested that cultivated Citrus species result from interspecific hybridization between four basic taxa (C. reticulata,C. maxima,C. medica and C. micrantha). However, the origin of most lemons and limes remains controversial or unknown. The aim of this study was to perform extended analyses of the diversity, genetic structure and origin of limes and lemons. The study was based on 133 Citrus accessions. It combined maternal phylogeny studies based on mitochondrial and chloroplastic markers, and nuclear structure analysis based on the evaluation of ploidy level and the use of 123 markers, including 73 basic taxa diagnostic single nucleotide polymorphism (SNP) and indel markers. The lime and lemon horticultural group appears to be highly polymorphic, with diploid, triploid and tetraploid varieties, and to result from many independent reticulation events which defined the sub-groups. Maternal phylogeny involves four cytoplasmic types out of the six encountered in the Citrus genus. All lime and lemon accessions were highly heterozygous, with interspecific admixture of two, three and even the four ancestral taxa genomes. Molecular polymorphism between varieties of the same sub-group was very low. Citrus medica contributed to all limes and lemons and was the direct male parent for the main sub-groups in combination with C. micrantha or close papeda species (for C. aurata, C. excelsa, C. macrophylla and C. aurantifolia--'Mexican' lime types of Tanaka's taxa), C. reticulata(for C. limonia, C. karna and C. jambhiri varieties of Tanaka's taxa, including popular citrus rootstocks such as 'Rangpur' lime, 'Volkamer' and 'Rough' lemons), C. aurantium (for C. limetta and C. limon--yellow lemon types--varieties of Tanaka's taxa) or the C. maxima × C. reticulate hybrid (for C. limettioides--'Palestine sweet' lime types--and C. meyeri). Among triploid limes, C. latifolia accessions ('Tahiti' and 'Persian' lime types) result from the fertilization of a haploid ovule of C. limon by a diploid gamete of C. aurantifolia, while C. aurantifolia triploid accessions ('Tanepao' lime types and 'Madagascar' lemon) probably result from an interspecific backcross (a diploid ovule of C. aurantifolia fertilized by C. medica). As limes and lemons were vegetatively propagated (apomixis, horticultural practices) the intra-sub-group phenotypic diversity results from asexual variations.

Nuclear species-diagnostic SNP markers mined from 454 amplicon sequencing reveal admixture genomic structure of modern citrus varieties.

Most cultivated Citrus species originated from interspecific hybridisation between four ancestral taxa (C. reticulata, C. maxima, C. medica, and C. micrantha) with limited further interspecific recombination due to vegetative propagation. This evolution resulted in admixture genomes with frequent interspecific heterozygosity. Moreover, a major part of the phenotypic diversity of edible citrus results from the initial differentiation between these taxa. Deciphering the phylogenomic structure of citrus germplasm is therefore essential for an efficient utilization of citrus biodiversity in breeding schemes. The objective of this work was to develop a set of species-diagnostic single nucleotide polymorphism (SNP) markers for the four Citrus ancestral taxa covering the nine chromosomes, and to use these markers to infer the phylogenomic structure of secondary species and modern cultivars. Species-diagnostic SNPs were mined from 454 amplicon sequencing of 57 gene fragments from 26 genotypes of the four basic taxa. Of the 1,053 SNPs mined from 28,507 kb sequence, 273 were found to be highly diagnostic for a single basic taxon. Species-diagnostic SNP markers (105) were used to analyse the admixture structure of varieties and rootstocks. This revealed C. maxima introgressions in most of the old and in all recent selections of mandarins, and suggested that C. reticulata × C. maxima reticulation and introgression processes were important in edible mandarin domestication. The large range of phylogenomic constitutions between C. reticulata and C. maxima revealed in mandarins, tangelos, tangors, sweet oranges, sour oranges, grapefruits, and orangelos is favourable for genetic association studies based on phylogenomic structures of the germplasm. Inferred admixture structures were in agreement with previous hypotheses regarding the origin of several secondary species and also revealed the probable origin of several acid citrus varieties. The developed species-diagnostic SNP marker set will be useful for systematic estimation of admixture structure of citrus germplasm and for diverse genetic studies.

A Phylogenetic Analysis of 34 Chloroplast Genomes Elucidates the Relationships between Wild and Domestic Species within the GenusCitrus

Citrus genus includes some of the most important cultivated fruit trees worldwide. Despite being extensively studied because of its commercial relevance, the origin of cultivated citrus species and the history of its domestication still remain an open question. Here, we present a phylogenetic analysis of the chloroplast genomes of 34 citrus genotypes which constitutes the most comprehensive and detailed study to date on the evolution and variability of the genus Citrus. A statistical model was used to estimate divergence times between the major citrus groups. Additionally, a complete map of the variability across the genome of different citrus species was produced, including single nucleotide variants, heteroplasmic positions, indels (insertions and deletions), and large structural variants. The distribution of all these variants provided further independent support to the phylogeny obtained. An unexpected finding was the high level of heteroplasmy found in several of the analyzed genomes. The use of the complete chloroplast DNA not only paves the way for a better understanding of the phylogenetic relationships within the Citrus genus but also provides original insights into other elusive evolutionary processes, such as chloroplast inheritance, heteroplasmy, and gene selection.

NEW INSIGHTS ON LIMES AND LEMONS ORIGIN FROM NUCLEAR AND CYTOPLASMIC MARKERS GENOTYPING AND TARGETED NUCLEAR GENE SEQUENCING

It is believed that Citrus medica, C. maxima, C. reticulate and C. micrantha have generated all cultivated citrus species. Depending on the classification, lemons and limes are classified either into two species, C. limon and C. aurantifolia (Swingle and Reece, 1967) or into more than 30 (Tanaka, 1977). In order to study the molecular phylogeny of this citrus group, we analyzed 23 targeted sequenced nuclear genes and used three mitochondrial and five chloroplastic markers for 15 lemons and limes compared with representatives of the four basic taxa. We observed three main groups, each one derived from direct interspecific hybridizations: (1) the Mexican lime group (C. aurantifolia), including C. macrophylla, arising from hybridizations between papeda (C. micrantha) and citron (C. medica); (2) the yellow lemon group (C. limon) that are hybrids between sour orange (C. aurantium, which is believed to be a hybrid between C. maxima and C. reticulata) and citron; and (3) a rootstock lemon/lime group (Rough lemon and Rangpur lime) that are hybrids between an acid mandarin and a citron. We also identified different probable backcrosses and genotypes with more complex origin. None of the analyzed limes and lemons shared the C. medica cytoplasm, while this taxon is the common nuclear contributor of all limes and lemons. Limes and lemons appear to be a very complex citrus varietal group with the contribution of the 4 basic taxa. Neither the Swingle and Reece classification nor the Tanaka fit with the genetic evidence. (Resume d'auteur)

Indian citrus ringspot virus의 ELISA 진단 시스템 구축

Indian citrus ring spot virus (ICRSV) is known to cause a serious disease to citrus, especially to Kinnow mandarin, the popular cultivated citrus species in India. In this study, we developed diagnostic systems based on enzyme-linked immunosorbent assay (ELISA). In order to generate antibodies against ICRSV coat protein, we overexpressed the coat protein in Escherichia coli using the pET15b expression vector containing an optimized ICRSV coat protein gene. The recombinant ICRSV coat protein was overexpressed as soluble form at upon IPTG induction. The protein was purified to 95% in purity by Ni-NTA column chromatography. The purified protein was immunized to rabbit for the generation of polyclonal antibody (PAb). The PAb showed a specific immunoreaction to recombinant ICRSV coat protein in western blot analysis and ELISA. Diluted rabbit antisera (10,000 fold) could detect less than 10 ng and 5 ng of the target protein in western blot and ELISA analysis, respectively.

Multilocus SNPs analysis allows phylogenetic assignation of DNA fragments to decipher the interspecific mosaic genome structure of cultivated citrus

All current studies seem to support the theory that four basic taxa (C. medica, C. maxima, C. reticulata and C. micrantha) have generated all cultivated Citrus species. It is supposed that the genomes of most of the modern Citrus cultivars, vegetatively propagated, are interspecific mosaic of large DNA fragments issued from a limited number of inter-specific meiosis. In the present work we analyse how multilocus study of closely linked SNPs allows a phylogenetic assignation of DNA fragments of the main cultivated species. Genomic fragments of 25 genes dispersed in the different chromosomes covering more than 12,5 Kb were amplified by PCR and sequenced (Sanger) for 24 accessions representative of 10 species. Moreover we checked the potential of parallel pyrosequencing (454 Roche) for direct multilocus haplotyping of heterozygous genotypes. Amplified fragments from 7 genes in 8 genotypes were obtained by using an original new method based on universal primers. C. clementina (Clementine) was used as model for secondary species. Citrus reticulata was the most polymorph basic taxa with an average of 4.2 SNPs/kb. The average differentiation between the basic taxa was about 20 SNPs/kb. For each amplified gene fragment, this polymorphism was enough for unambiguous multilocus differentiation of the basic species and assignation of a phylogenetic origin for the secondary species. A preliminary reconstitution of phylogenetic structure of chromosome 3 is proposed for sweet orange, sour orange, grapefruit, lemon and lime. Consensus haplotype sequences were successfully obtained from 454 sequencing with genotype sequence in total agreement with Sanger control. Each haplotype sequence of Clementine was univocally assigned to one of the haplotype clusters of the basic taxa. Phylogenetic origin of specific DNA fragments can be assigned from multilocus analysis of closely linked SNPs. Multilocus haplotyping by parallel sequencing of individual DNA molecule will be a very powerful tool to decipher the interspecific mosaic genome structure of cultivated citrus. (Resume d'auteur)

Intensity of attack caused by Mediterranean fruit fly Ceratitis capitata Wiedem. (Diptera, Tephritidae) on mandarin along the Montenegrin seacoast

Along the Montenegrin seacoast, all cultivated citrus species (mandarin, orange, lemon, grapefruit), fig, loquat and ziziphus were detected as host plants of the Ceratitis capitata Wiedem. Among those found, in economic sense, the most important host plant is mandarin Unsiu. Intensity of the attack were monitored on mandarin in 2003 and 2004 in localities Baosici, Lastva Grbaljska and Bar, and was determined as higher in 2003. The first symptoms of attack were detected, depending on locality, from middle of September until beginning of October, one to two months earlier then in 2004. The maximum number of larvae per fruit were detected in October and November (average from 13.44?0.16 to 22.82?0.13). Time of the first symptoms appearence on mandarin in September and October 2003 and in November 2004, indicate on crucial importance of alternative host plants (figs and loquat) for reproduction and increasing of C. capitata population, because later during a season its intensity of attack on mandarin, depend largely on their presence.

Volatile Organic Components of Fresh Leaves as Indicators of Indigenous and Cultivated Citrus Species in Taiwan

The volatile components of fresh leaves from 15 citrus species were investigated by headspace SPME with a GC-MS analysis. Three indigenous Taiwan citrus species, Citrus taiwanica, C. tachibana and C. depressa, were the major subjects. Eighty volatile organic compounds were detected as indicators of the genetic relationship. Linalool was the most abundant compound, and citronellal, geranial, neral, limonene and trans-beta-ocimene were the major volatile compounds in fresh leaves. Linalool (56.37%) and myrcene (7.21%) were predominant in C. tawanica. An aldehyde-rich profile with citronellal (24.54%) contributed most to the aroma of leaves in C. tachibana, while Citrus depressa exhibited a high linalool/citronellal composition (23.56%/12.51%). The qualitative and quantitative patterns of the volatiles revealed that C. taiwanica was linked with sour orange, and either C. tachibana or C. depressa belonged to the mandarin group with C. tankan. Dendrograms also showed that the volatile patterns were related to the genetic classification.