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Machine learning, a subset of artificial intelligence, is revolutionizing agriculture by enabling enhanced crop monitoring, disease detection, and yield prediction. Its application extends to precision farming, where it aids in optimizing irrigation, fertilization, and harvesting by analyzing large datasets from mobile phones, sensors and satellite images. It has played a crucial role in identifying and categorizing different types of plants. Specifically, Convolutional Neural Networks (CNN), a deep learning technology within ML, are employed to classify plant species based on images. In the present research work, we have employed machine learning for the classification of citrus species using Convolutional Neural Networks (CNNs). A dataset has been developed with images of fruits and leaves from ten citrus species. We have utilized transfer learning with architectures like Mobile Net, Alex Net, and Goog Le Net. The study demonstrates that combining multiple plant components in CNN analysis improves classification accuracy, with leaves providing more reliable results than fruits. This approach signifies a major advancement in agricultural technology, allowing for more precise and efficient farming practices. The findings indicate that expanding the dataset and incorporating more plant structures could further refine these models. This research highlights the potential of machine learning in agriculture, particularly in enhancing species classification, which is crucial for sustainable and productive farming. [ABSTRACT FROM AUTHOR]

Minerals are reported to dominate the electrical properties of honey and indicate its botanical and geographical origins. In this study, Electrochemical Impedance Spectroscopy (EIS) was used to assess the relation between mineral elements, electrical properties and botanical origin using three honey varieties - Citrus sp., Eucalyptus sp., and Erica sp. These varieties are identified through pollen analysis and market labelling. Flame atomic absorption and emission spectroscopies were used to quantify the concentrations of eight elements (potassium, sodium, calcium, magnesium, manganese, zinc, copper, and iron). Among all the mineral elements, potassium showed a consistent correlation with impedance. The potassium estimation in honey and standard solutions (calibration curve) had similar sensitivities of 153.43 nF/mM and 132.68 nF/mM, respectively. Additionally, the analysis revealed that potassium dominates the mineral composition, with the other species present in minimal quantities. The EIS technique showed high sensitivity to potassium and other ionisable species, making it possible to classify the botanical origin of these three honey types. The EIS technique proved to be both time and cost effective, yielding a classification rate higher than that achieved by analysing mineral composition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Citrus peels are rich in polymethoxylated flavones (PMFs), which have beneficial health and pharmacological properties. In this study, the profiles, variations, and biological activities of PMFs in the peel extracts of 27 Citrus varieties (eight species) native to China were investigated. UPLC-QTOF-MS/MS analysis revealed that mandarin accumulated more diversity and higher detectable PMF contents. Wangcangzhoupigan (ZPG) possessed the highest antioxidant capacity. Gailiangcheng (GLC) and Bingtangcheng (BTC), sweet oranges showed excellent inhibitory effects against pancreatic lipase and α-glucosidase, respectively. Most citrus extracts effectively inhibited the production of ROS and pro-inflammatory cytokines, while increasing the accumulation of anti-inflammatory cytokines. In addition, Limeng (LM), Cupig-oushigan (GSG), and Yanxiwanlu (YXWL) showed anti-proliferative effects against DU145 and PC3 cancer cells. This study provides a comprehensive PMF profile and biological activities of various citrus species and will benefit future functional citrus breeding practices aimed at designing plants rich in total or specific PMFs for health benefits., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A total of 145 accessions of the genus Citrus and related genera, maintained in the Conservation Garden for Citrus Germplasm at the Experimental Farm of Kindai University, Yuasa, Wakayama, Japan, were examined for their phylogenetic relationships. The present classification was conducted using an inter-retrotransposon amplified polymorphism (IRAP) method based on the insertion polymorphism of a retrotransposon, CIRE1, identified in C. sinensis. The objective of this study was to evaluate the applicability of the IRAP method for citrus classification. The constructed dendrogram showed that the 145 accessions and two outgroup species were successfully classified into five major clades. A large number of C. sinensis accessions were divided into three traditional groups, navel orange, sweet orange, and blood orange, almost corresponding to the sub-clades in the dendrogram. Several other accessions belonging to the same species, and also many hybrid cultivars from crossbreeding, were localized into the respective sub-clades or near positions in the dendrogram. Several unclassified accessions could also be located in the dendrogram, suggesting novel relationships with other accessions. It was concluded that the IRAP method based on CIRE1 insertion polymorphism was suitable for the classification of citrus from a molecular point of view.

Background: Natural medicines present a considerable analytical challenge due to their diverse botanical origins and complex multi-species composition. This inherent complexity complicates their rapid identification and analysis. Tangerine peel, a product of the Citrus species from the Rutaceae family, is widely used both as a culinary ingredient and in traditional Chinese medicine. It is classified into two primary types in China: Citri Reticulatae Pericarpium (CP) and Citri Reticulatae Pericarpium Viride (QP), differentiated by harvest time. A notable price disparity exists between CP and another variety, Citri reticulatae "Chachi" (GCP), with differences being based on the original variety., Methods: This study introduces an innovative method using portable miniature mass spectrometry for swift on-site analysis of QP, CP, and GCP, requiring less than a minute per sample. And combined with machine learning to differentiate the three types on site, the method was used to try to distinguish GCP from different storage years., Results: This novel method using portable miniature mass spectrometry for swift on-site analysis of tangerine peels enabled the characterization of 22 compounds in less than one minute per sample. The method simplifies sample processing and integrates machine learning to distinguish between the CP, QP, and GCP varieties. Moreover, a multiple-perceptron neural network model is further employed to specifically differentiate between CP and GCP, addressing the significant price gap between them., Conclusions: The entire analytical time of the method is about 1 minute, and samples can be analyzed on site, greatly reducing the cost of testing. Besides, this approach is versatile, operates independently of location and environmental conditions, and offers a valuable tool for assessing the quality of natural medicines., (© 2024 John Wiley & Sons Ltd.)

Aurantii Fructus Immaturus(AFI) is a traditional Chinese herbal medicine with multiple origins from Citrus aurantium and its legally cultivated variants. With advancements in agricultural biotechnology, many new cultivated varieties have sprung up,leading to an abundance of AFI adulterants and chaos in the herbal medicine markets. This study developed a specific identification method for AFI and its closely related adulterants by examining the appearance trait, content of extract, and multiple ingredients,involving indicators such as the ratio of pulp capsule to cross section diameter(Pc/Cs ratio), the content of extract, and the profile of 11 ingredients. The research finds that:(1) Pc/Cs ratio can conveniently identify adulterants such as Poncirus trifoliata, Ju, and Babagan from the genuine AFI.(2) The extract content can be used to identify adulterants originated from C. wilsonii with C. aurantium.(3) The contents of synephrine in all the samples were in accordance with the Chinese Pharmacopoeia except for the adulterants from P. trifoliata, C. wilsonii, C. aurantium 'Changshanhuyou' and orah mandarins. The synephrine content was high as 1. 40% in some C. sinensis varieties. The mass fraction of hesperidin was over 10. 00% in C. sinensis, while it was below 2. 50% in C. aurantium. C. aurantium contained high levels of naringin(3. 96%-15. 21%) and neo-hesperidin(9. 38%-21. 93%).(4) The compositions of adulterants from P. trifoliata and C. wilsonii were more similar to that of C. aurantium 'Daidai', but with significantly lower neo-hesperidin content(0. 03%-0. 14%) than that in C. aurantium, and they lacked hesperetin and tangeretin. C. maxima(originating from C. maxima) showed closer composition to Choucheng and hybrid originated from Citrus aurantium × Poncirus trifoliata, but had higher hesperidin content(3. 13%) than that in C. aurantium. Ju was closely related to C. sinensis and neither contained naringin nor neo-hesperidin. Hesperidins in Babagan and orah mandarins were similar to that in C. sinensis, with none containing rhoifolin. These quality indicators in combination can accurately distinguish between C. sinensis, C. aurantium, and their closely related adulterants(P. trifoliata, C. wilsonii, C. maxima, orah mandarins and C. reticulata), which are expected to provide a systematic method for quality control of AFI.

In allusion to the problems of citrus surface defect identification such as blurred edges, unclear images, more interference and difficulty in defect identification, surface defect identification of citrus based on KF-2D-Renyi and ABC-SVM was proposed in this paper. First, the method based on the dark channel prior (DCP) was used to defog the citrus images collected. Then, the firefly algorithm based on Kent chaos was used to optimize two-dimensional Renyi entropy threshold segmentation algorithm (2D-Renyi). The citrus surface defects were segmented, and the image features were extracted. Finally, the image feature vectors were input into the ABC-SVM classifier to determine the citrus defect types. We selected 8 kinds of citrus surface defects to carry on the experiment. In testing the segmentation algorithms, compared with the traditional threshold segmentation algorithms, the KF-2D-Renyi segmentation algorithm has a great improvement. The recognition rates for the defects whose features are obvious such as Sooty mould and Anthracnose could reach 100%. The recognition rates for the defects which are difficult to identify such as Thrips scar, Oleocellosis and Scale injury reached 95.18%, 96.37% and 98.43% respectively. In testing the classification algorithms, compared with the standard SVM classifier, the PSO-SVM classifier and the neural network classifiers, the average recognition rate of the ABC-SVM classifier reached 98.45%. The experimental results show that the method in this paper can effectively detect and classify citrus surface defects. [ABSTRACT FROM AUTHOR]

Citrus fruits are among the most economically important crops in the world. In the global market, the Citrus peel is often considered a byproduct but substitutes an important phenotypic characteristic of the fruit and a valuable source of essential oils, flavonoids, carotenoids, and phenolic acids with variable concentrations. The Mediterranean basin is a particularly dense area of autochthonous genotypes of Citrus that are known for being a source of healthy foods, which can be repertoires of valuable genes for molecular breeding with the focus on plant resistance and quality improvement. The scope of this study was to characterize and compare the main phenotypic parameters ( i.e. , peel thickness, fruit volume, and area) and levels of bioactive compounds in the peel of fruits from the local germplasm of Citrus in Greece, to assess their chemodiversity regarding their polyphenolic, volatile, and carotenoid profiles. A targeted liquid chromatographic approach revealed hesperidin, tangeretin, narirutin, eriocitrin, and quercetin glycosides as the major polyphenolic compounds identified in orange, lemon, and mandarin peels. The content of tangeretin and narirutin followed the tendency mandarin > orange > lemon. Eriocitrin was a predominant metabolite of lemon peel, following its identification in lower amounts in mandarin and at least in the orange peel. For these citrus-specific metabolites, high intra- but also interspecies chemodiversity was monitored. Significant diversity was found in the essential oil content, which varied between 1.2 and 3% in orange, 0.2 and 1.4% in mandarin, and 0.9 and 1.9% in lemon peel. Limonene was the predominant compound in all Citrus species peel essential oils, ranging between 88 and 93% among the orange, 64 and 93% in mandarin, and 55 and 63% in lemon cultivars. Carotenoid analysis revealed different compositions among the Citrus species and accessions studied, with β-cryptoxanthin being the most predominant metabolite. This large-scale metabolic investigation will enhance the knowledge of Citrus peel secondary metabolite chemodiversity supported by the ample availability of Citrus genetic resources to further expand their exploitation in future breeding programs and potential applications in the global functional food and pharmaceutical industries.

The Gibberellic Acid Stimulated Arabidopsis (GASA) proteins were investigated in the study to help understand their possible roles in fruit trees, particularly in Citrus. A total of 18 CcGASA proteins were identified and characterized in Citrus clementina via a genome-wide approach. It was shown that the CcGASA proteins structurally shared a conserved GASA domain but varied considerably in primary sequences and motif compositions. Thus, they could be classified into three major phylogenetic groups, G1~G3, and two groups, G1 and G3 could be further classified into subgroups. The cis- elements on all CcGASA promoters were identified and categorized, and the associated transcription factors were predicted. In addition, the possible interactions between the CcGASA proteins and other proteins were predicted. All the clues suggested that these genes should be involved in defense against biotic and abiotic stresses and in growth and development. The notion was further supported by gene expression analysis that showed these genes were more or less responsive to the treatments of plant hormones (GA 3 , SA, ABA and IAA), and infections of citrus canker pathogen Xanthomonas citri. It was noted that both the segmental and the tandem duplications had played a role in the expansion of the CcGASA gene family in Citrus. Our results showed that the members of the CcGASA gene family should have structurally and functionally diverged to different degrees, and hence, the representative group members should be individually investigated to dissect their specific roles., (© 2021. The Author(s).)

The genus Citrus contains a vast range of antioxidant metabolites, dietary metabolites, and antioxidant polyphenols that protect plants from unfavorable environmental conditions, enhance their tolerance to abiotic and biotic stresses, and possess multiple health-promoting effects in humans. This review summarizes various antioxidant metabolites such as organic acids, amino acids, alkaloids, fatty acids, carotenoids, ascorbic acid, tocopherols, terpenoids, hydroxycinnamic acids, flavonoids, and anthocyanins that are distributed in different citrus species. Among these antioxidant metabolites, flavonoids are abundantly present in primitive, wild, and cultivated citrus species and possess the highest antioxidant activity. We demonstrate that the primitive and wild citrus species (e.g., Atalantia buxifolia and C. latipes ) have a high level of antioxidant metabolites and are tolerant to various abiotic and biotic stresses compared with cultivated citrus species (e.g., C. sinensis and C. reticulata ). Additionally, we highlight the potential usage of citrus wastes (rag, seeds, fruit peels, etc.) and the health-promoting properties of citrus metabolites. Furthermore, we summarize the genes that are involved in the biosynthesis of antioxidant metabolites in different citrus species. We speculate that the genome-engineering technologies should be used to confirm the functions of candidate genes that are responsible for the accumulation of antioxidant metabolites, which will serve as an alternative tool to breed citrus cultivars with increased antioxidant metabolites.

Citrus secondary metabolites, such as terpene compounds, are very important for human health due to their bioactivity including anti-inflammatory, anti-cancer, and antioxidant effects. In this work, for the first time, the volatile chemical composition of peels and juices from four different Citrus species ( C. junos , Citrus × aurantium , C. aurantium 'Bizzarria' and C. medica 'Florentina', commonly known as Yuzu jeune, Oni Yuzu, Bizzarria orange and Florence cedar, respectively) was investigated by Solid-Phase Microextraction-Gas Chromatography/Mass Spectrometry (SPME-GC/MS) technique and the antiradical activity was also examined. The results showed that limonene and γ-terpinene were the main volatile substances detected both in the juices and in the peels, followed by other minority compounds responsible for the phyto-complex of the unique aromas which characterize each individual analyzed Citrus species. Principal component analysis (PCA), performed on volatile compounds, showed both some correlation as well as a clear separation between the juice and the peel of each species. Among them, Oni Yuzu juice was found to be the richest in total polyphenols and flavonoids while its capacity to scavenge ABTS •+ and DPPH • radicals was similar to that of Yuzu Jeune and Bizzarria orange.

The origin and dispersal of cultivated and wild mandarin and related citrus are poorly understood. Here, comparative genome analysis of 69 new east Asian genomes and other mainland Asian citrus reveals a previously unrecognized wild sexual species native to the Ryukyu Islands: C. ryukyuensis sp. nov. The taxonomic complexity of east Asian mandarins then collapses to a satisfying simplicity, accounting for tachibana, shiikuwasha, and other traditional Ryukyuan mandarin types as homoploid hybrid species formed by combining C. ryukyuensis with various mainland mandarins. These hybrid species reproduce clonally by apomictic seed, a trait shared with oranges, grapefruits, lemons and many cultivated mandarins. We trace the origin of apomixis alleles in citrus to mangshanyeju wild mandarins, which played a central role in citrus domestication via adaptive wild introgression. Our results provide a coherent biogeographic framework for understanding the diversity and domestication of mandarin-type citrus through speciation, admixture, and rapid diffusion of apomictic reproduction., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Nanfeng mandarins ( Citrus reticulata Blanco cv. Kinokuni), Xunwu mandarins ( Citrus reticulata Blanco), Yangshuo kumquats ( Citrus japonica Thunb) and physiologically dropped navel oranges ( Citrus sinensis Osbeck cv. Newhall) were used as materials to extract peel essential oils (EOs) via hydrodistillation. The chemical composition, and antibacterial and antioxidant activities of the EOs were investigated. GC-MS analysis showed that monoterpene hydrocarbons were the major components and limonene was the predominate compound for all citrus EOs. The antibacterial testing of EOs against five different bacteria ( Bacillus subtilis , Staphylococcus aureus , Escherichia coli , Pseudomonas aeruginosa and Salmonella typhi murium ) was carried out using the filter paper method and the broth microdilution method. Kumquat EO had the best inhibitory effect on B . subtilis , E . coli and S . typhi murium with MIC (minimum inhibitory concentration) values of 1.56, 1.56 and 6.25 µL/mL, respectively. All citrus EOs showed the antioxidant activity of scavenging DPPH and ABTS free radicals in a dose-dependent manner. Nanfeng mandarin EO presented the best antioxidant activity, with IC 50 values of 15.20 mg/mL for the DPPH assay and 0.80 mg/mL for the ABTS assay. The results also showed that the antibacterial activities of EOs might not be related to their antioxidant activities.

A prototype dual-detection headspace-gas chromatography-mass spectrometry-ion mobility spectrometry (HS-GC-MS-IMS) system was used for the analysis of the volatile profile of 47 Citrus juices including grapefruit, blood orange, and common sweet orange juices without requiring any sample pretreatment. Next to reduced measurement times, substance identification could be improved substantially in case of co-elution by considering the characteristic drift times and m / z ratios obtained by IMS and MS. To discriminate the volatile profiles of the different juice types, extensive data analysis was performed with both datasets, respectively. By principal component analysis (PCA), a distinct separation between grapefruit and orange juices was observed. While in the IMS data grapefruit juices not from fruit juice concentrate could be separated from grapefruit juices reconstituted from fruit juice concentrate, in the MS data, the blood orange juices could be differentiated from the orange juices. This observation leads to the assumption that the IMS and MS data contain different information about the composition of the volatile profile. Subsequently, linear discriminant analysis (LDA), support vector machines (SVM), and the k -nearest-neighbor ( k NN) algorithm were applied to the PCA data as supervised classification methods. Best results were obtained by LDA after repeated cross-validation for both datasets, with an overall classification and prediction ability of 96.9 and 91.5% for the IMS data and 94.5 and 87.9% for the MS data, respectively, which confirms the results obtained by PCA. Additional data fusion could not generally improve the model prediction ability compared to the single data, but rather for certain juice classes. Consequently, depending on the juice class, the most suitable dataset should be considered for the prediction of the class membership. This volatilomic approach based on the dual detection by HS-GC-MS-IMS and machine learning tools represent a simple and promising alternative for future authenticity control of Citrus juices.

Citrus fruit are sensitive to chilling injury (CI) during cold storage, a peel disorder that causes economic losses. C-repeat binding factors (CBFs) are related to cold acclimation and tolerance in different plants. To explore the role of Citrus CBFs in fruit response to cold, an in silico study was performed, revealing three genes ( CBF1 , CBF2 , and CBF3 ) whose expression in CI sensitive and tolerant cultivars was followed. Major changes occurred at the early stages of cold exposure (1-5 d). Interestingly, CBF1 was the most stimulated gene in the peel of CI-tolerant cultivars (Lisbon lemon, Star Ruby grapefruit, and Navelina orange), remaining unaltered in sensitive cultivars (Meyer lemon, Marsh grapefruit, and Salustiana orange). Results suggest a positive association of CBF1 expression with cold tolerance in Citrus cultivars (except for mandarins), whereas the expression of CBF2 or CBF3 genes did not reveal a clear relationship with the susceptibility to CI. Light avoidance during fruit growth reduced postharvest CI in most sensitive cultivars, associated with a rapid and transient enhance in the expression of the three CBFs. Results suggest that CBFs-dependent pathways mediate at least part of the cold tolerance responses in sensitive Citrus , indicating that CBF1 participates in the natural tolerance to CI.

Most satsuma mandarin (Citrus unshiu Marc.) cultivars are difficult to identify in the seedling stage based only on morphological traits. Therefore, simple polymerase chain reaction (PCR)-based single-nucleotide polymorphism (SNP) markers were developed to specifically and rapidly distinguish the 'Haryejosaeng' cultivar, which is generally supplied to breeders of other satsuma mandarin cultivars. SNP markers were verified using high-resolution melt (HRM)-specific primers. PCR was performed to distinguish 'Haryejosaeng' from eight other satsuma mandarin cultivars using six SNP markers (P1-P6) specific for 'Haryejosaeng', with one negative control SNP primer pair. The best results were obtained using three SNP markers (P1, P2, and P5). In the multiplex PCR, markers P1, P2, and P5 yielded 165-, 150-, and 526-base pair amplicons, respectively, in 'Haryejosaeng', distinguishing it from other satsuma mandarin cultivars. The selected SNP markers were validated by HRM with HRM-specific primers. The multiplex PCR with P1/P5 and P2/P5 also identified 'Haryejosaeng' obtained from a farm growing 17 different cultivars of satsuma mandarin. Specific SNP molecular markers were determined for accurately identifying the 'Haryejosaeng' cultivar by multiplex PCR to save the time and costs associated with its supply to breeders of satsuma mandarin.

Drought stress as one of the most devastating abiotic stresses affects agricultural and horticultural productivity in many parts of the world. The application of melatonin can be considered as a promising approach for alleviating the negative impact of drought stress. Modeling of morphological responses to drought stress can be helpful to predict the optimal condition for improving plant productivity. The objective of the current study is modeling and predicting morphological responses (leaf length, number of leaves/plants, crown diameter, plant height, and internode length) of citrus to drought stress, based on four input variables including melatonin concentrations, days after applying treatments, citrus species, and level of drought stress, using different Artificial Neural Networks (ANNs) including Generalized Regression Neural Network (GRNN), Radial basis function (RBF), and Multilayer Perceptron (MLP). The results indicated a higher accuracy of GRNN as compared to RBF and MLP. The great accordance between the experimental and predicted data of morphological responses for both training and testing processes support the excellent efficiency of developed GRNN models. Also, GRNN was connected to Non-dominated Sorting Genetic Algorithm-II (NSGA-II) to optimize input variables for obtaining the best morphological responses. Generally, the validation experiment showed that ANN-NSGA-II can be considered as a promising and reliable computational tool for studying and predicting plant morphological and physiological responses to drought stress., Competing Interests: The authors have declared that no competing interests exist.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the only natural vector of bacteria responsible for Huanglongbing (HLB), a worldwide destructive disease of citrus. ACP reproduces and develops only on the young leaves of its rutaceous host plants. Olfactory stimuli emitted by young leaves may play an important role in ACP control and HLB detection. In this study, volatile organic compounds (VOCs) from healthy and HLB-infected young leaves of navel orange and pomelo were analyzed by headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS). A total of 36 compounds (including dimers or polymers) were identified and quantified from orange and 10 from pomelo leaves. Some compounds showed significant differences in signal intensity between healthy and HLB-infected leaves and may constitute possible indicators for HLB infection. Principal component analysis (PCA) clearly discriminated healthy and HLB-infected leaves in both orange and pomelo. HS-GC-IMS was an effective method to identify VOCs from leaves. This study may help develop new methods for detection of HLB or find new attractants or repellents of ACP for prevention of HLB.

Background: Ichang Papeda (Citrus cavaleriei H.Lév. ex Cavalerie) is a wild and endangered species of NE India that requires urgent preservation of its genetic resources., Objective: To characterize the storage physiology of the seeds and to cryopreserve the embryo and embryonic axis (EA)., Materials and Methods: The morphology and storage physiology of the seeds were determined, and the cryopreservation of embryos and EA attempted using various techniques (viz., air desiccation-freezing, vitrification and encapsulation-dehydration)., Results: Weighing up to 4 g, seeds of Citrus cavaleriei are the largest known in the genus. Based on estimates using the seed coat ratio - seed mass (SCR-SM) model there was a very high probability of seed desiccation intolerance, which was validated physiologically; seeds lost vigour on drying below 30% moisture content (MC) and no seeds germinating after drying to <12% MC. Embryos and EAs could be air dried to 25-30% MC and cryopreserved with c. 50% survival. In contrast, EA optimally exposed to PVS2 (20 min) or encapsulated, sucrose pretreated (0.5 M, 24 h) and dehydrated (6 h) had c. 40% survival after cryopreservation., Conclusion: Citrus cavaleriei produces large, recalcitrant seeds that can, nevertheless, be cryopreserved as embryos or isolated EA after air drying to c. 25-30% MC; encapsulation-dehydration and vitrification provide alternative options for the cryopreservation of EA.

Background: The fluorescence sensing method has been increasingly applied in food quality control because it is fast and sensitive. However, its application in quality evaluation is challenging. Using Citri Reticulatae Pericarpium (CRP; dried mandarin orange peel) as an example, we developed a simple and low-cost fluorescence sensing strategy based on nanoparticles combined with spectral splicing and chemometrics for quality evaluation. This method can recognize Citrus reticulata 'Chachi' (CRC) from other CRP cultivars and further identify the storage year., Results: Nanogold particles and cadmium telluride quantum dots were selected as nanosensors and mixed with aqueous extracts of CRP separately to produce fluorescence quenching spectra. Then, a simple spectral splicing procedure was applied to obtain spliced spectra comprising different combinations of the self-fluorescence and fluorescence quenching spectra of CRP samples. With the aid of partial least-squares discriminant analysis, the new strategy achieved recognition rates of 100% in distinguishing CRC samples from other CRP samples, as well as recognition rates of 100% for the training set and 98.04% for the prediction set in the discrimination of the storage year of CRC. The recognition mechanism is dominated by interactions between the nanoparticles and the fluorescent components in the CRP samples, but other components also have concurrent effects., Conclusions: This novel fluorescence sensing strategy not only provides a new tool for the quality evaluation of CRC but also has good prospects for the authentication and traceability of other foods and herbs. Crucially, the developed method is convenient, simple and effective. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Background: Anthocyanin pigmentation characterizes a number of tissues of Citrus and its relatives. The gain and loss of pigmentation is intriguing and is inherited variously among species., Methods: Citrus germplasm was used to investigate the anthocyanin pigmentation of tissues never before considered, including stamen, style and stigma, and of young leaves, petals, rind and flesh of 28 genotypes belonging to 14 species. Citrus genotypes encompassed citron, lemon, sweet orange, lime, and Citrus relatives included Microcitrus , Murraya , and Severinia . A relative qRT-PCR analysis was carried out on the structural and regulatory genes: phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3'-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), uridine diphosphate glucose flavonoid glucosyl-transferase (UFGT), glutathione S-transferase (GST), Ruby and Noemi. Image analysis and a genomic approach were employed to evaluate how the red pigmentation is inherited among tissues and species., Results: Pigmentation of young leaves and petals is specific to citron and its hybrids. Ruby controls the pigmentation of petals, but not of leaves. The red color of the rind and flesh is a trait that particularly characterizes a diversity of sweet oranges, citron hybrids and Citrus relatives. Color expression depends on external factors and also on developmental stage. The coloration of stamen and style is citron-specific, while a red stigma is exclusive to Moro orange and its hybrids., Conclusion: It is hypothesized that there is a relationship among Citrus species and genes controlling anthocyanin pigmentation.

Mandarins (or tangerines) are mainly consumed as fresh fruits due to the ease of peeling and desirable flavor. Sweetness, acidity, and flavor of mandarin are the most important criteria for consumer preference. The objective of this study was to evaluate the effects of harvest date on sensory and chemical components of four mandarin cultivars (Murcott, 411, Temple, and 'LB8-9' Sugar Belle®). Volatiles were extracted from the headspace of juice samples with solid phase microextraction (SPME) and analyzed using gas chromatography-mass spectrometry (GC-MS). The optimum harvest window for eating quality of 411 was late January to mid-February (soluble solids content [SSC]/titratable acidity [TA]: 11.3 to 14.0), Sugar Belle® fruits were best tasted when harvested from mid- to end of January (SSC/TA: 14.1 to 16.1), and February was the best month for harvesting Murcott (SSC/TA: 13.10 to 18.0) and Temple (SSC/TA:10.3 to 12.50). Sensory perception of sweetness, ripeness, and juiciness increased as SSC/TA increased while sourness and bitterness decreased. Pumpkin flavor, an indicator of overripe fruit, was mainly noticed late in the season. Tangerine flavor tended to decrease, whereas fruity-noncitrus flavor tended to increase with fruit maturity. Monoterpenes were the most abundant volatiles and tended to decrease with fruit maturity, whereas alcohols, esters, and aldehydes increase. Aldehydes, esters, and alcohols were positively correlated with sweetness, ripeness, juiciness, and fruity characteristics, and negatively with sourness and bitterness. On the other hand, monoterpenes were positively correlated with bitterness and tangerine flavor, and negatively correlated with sweetness and fruity-noncitrus flavor. The highest number of esters was found in Temple, whereas Murcott and 411 were high in aldehydes., (© 2020 Institute of Food Technologists®.)

Short interspersed nuclear elements (SINEs) are non-autonomous retrotransposons that are highly abundant, but not well annotated, in plant genomes. In this study, we identified 41,573 copies of SINEs in seven citrus genomes, including 11,275 full-length copies. The citrus SINEs were distributed among 12 families, with an average full-length rate of 0.27, and were dispersed throughout the chromosomes, preferentially in AT-rich areas. Approximately 18.4% of citrus SINEs were found in close proximity (≤1 kb upstream) to genes, indicating a significant enrichment of SINEs in promoter regions. Citrus SINEs promote gene and genome evolution by offering exons as well as splice sites and start and stop codons, creating novel genes and forming tandem and dispersed repeat structures. Comparative analysis of unique homologous SINE-containing loci (HSCLs) revealed chromosome rearrangements in sweet orange, pummelo, and mandarin, suggesting that unique HSCLs might be valuable for understanding chromosomal abnormalities. This study of SINEs provides us with new perspectives and new avenues by which to understand the evolution of citrus genes and genomes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

In this study, we aim to determine the chemical constituents of six Chinese medicinal materials from the Citrus genus using high-performance liquid chromatography and high-resolution mass spectrometry. Eight flavonoids and one coumarin were identified and further quantified as marker substances by high-performance liquid chromatography method. The separation was performed on an Agilent TC-C 18 column with 0.1% formic acid and acetonitrile as the mobile phase under gradient elution. The analytical method was fully validated in terms of linearity, sensitivity, intra- and inter-day precision and repeatability, limit of detection, limit of quantitation, and recovery. It was subsequently applied to evaluate the quality of 103 batches of the Chinese medicinal materials from the Citrus genus. In addition, the principal constituent analysis was used to compare the samples of different species from the Citrus genus leading to successful classification of the samples in accordance with their origins. It was found that the contents of nine constituents varied greatly in different ripening stages and varieties of the samples from the Citrus genus. In addition, neoeriocitrin and 5,7-dimethoxycoumarin were determined as two unique constituents of 'Zhiqiao' and 'Foshou', respectively. In conclusion, this study provides a chemical basis for quality control of Chinese medicinal materials from the Citrus genus., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aim of the study was to compare the mineral content between the peel and the pulp of citrus fruits and to determine which citrus fruit, among orange (Citrus sinensis), pomelo (Citrus maxima), mandarin (Citrus reticulata Blanco), lemon (Citrus limon), key lime (Citrus aurantifolia), and red, yellow, or green grapefruit (Citrus paradisi), is the richest in minerals. The research material consisted of fresh citrus fruits belonging to the genus Citrus L in the family Rutaceae. The fruits were purchased at a supermarket at one time. To prepare laboratory samples, each fruit was cut in half, and one half was homogenized, treating the sample as a whole (peel + flesh), while the other half was peeled and the pulp (F) and peel (P) were homogenized separately. To determine the content of minerals (Na + , K + , Ca +2 , Mg +2 , Fe +2 , Zn +2 , Cu +2 , Mn +2 , and Se +2 ), the samples were mineralized and analyzed using an Analytik Jena PlasmaQuant PQ 9000 inductively coupled plasma optical emission spectrometer. The content of macro- and micronutrients in the peel of most of the fruits far exceeded their quantity in the pulp. Oranges and pomelos are the fruits richest in iron and copper, so they could be recommended in cases such as hemoglobin production disorders resulting from a deficiency of these elements. Oranges can additionally enrich the body with potassium, phosphorus, and manganese, while lime can be a source of calcium, zinc, sodium, and especially potassium. It should also be noted that all citrus fruits are a very valuable source of potassium, which is needed to ensure the water and electrolyte balance.

Speciation of the genus Citrus from a common ancestor has recently been established to begin ∼8 Ma during the late Miocene, a period of major climatic alterations. Here, we report the changes in activity of Citrus LTR retrotransposons during the process of diversification that gave rise to the current Citrus species. To reach this goal, we analyzed four pure species that diverged early during Citrus speciation, three recent admixtures derived from those species and an outgroup of the Citrus clade. More than 30,000 retrotransposons were grouped in ten linages. Estimations of LTR insertion times revealed that retrotransposon activity followed a species-specific pattern of change that could be ascribed to one of three different models. In some genomes, the expected pattern of gradual transposon accumulation was suddenly arrested during the radiation of the ancestor that gave birth to the current Citrus species. The individualized analyses of retrotransposon lineages showed that in each and every species studied, not all lineages follow the general pattern of the species itself. For instance, in most of the genomes, the retrotransposon activity of elements from the SIRE lineage reached its highest level just before Citrus speciation, while for Retrofit elements, it has been steadily growing. Based on these observations, we propose that Citrus retrotransposons may respond to stressful conditions driving speciation as a part of the genetic response involved in adaptation. This proposal implies that the evolving conditions of each species interact with the internal regulatory mechanisms of the genome controlling the proliferation of mobile elements., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Citrus fruits have been introduced to the Mediterranean area from Asia for centuries and spontaneous crosses have generated several hybrid forms, some of which have had agricultural or industrial success while others have remained niche food or ornamental products, or have disappeared. Pompia (C. medica tuberosa Risso & Poiteau) is an old endemic citrus fruit from Sardinia of unknown genetic origin. Initial phenotypic and molecular characterizations revealed a high degree of similarity with lemon (C. limon (L.) Burm.) and citron (C. medica L.). To identify the ancestors of Pompia, 70 citrus species of the Citrus genus were genotyped with 36 codominant molecular markers (SSR and InDel) of nuclear and cytoplasmic genomes. Diversity analysis and allelic comparisons between each citrus species at each locus indicated that Pompia resembles lemon and limonette of Marrakech, i.e. the result of a cross between sour orange (C. aurantium L.) and citron, where citron was the pollinator. Two Italian citron varieties were identified as potential male parents, i.e. Diamante and Common Poncire. However, we were unable to differentiate varieties of sour oranges because varietal diversification in this horticultural group resulted from DNA sequence variations that SSR or InDel markers could not reveal. Rhob el Arsa and Poncire de Collioure were found to be two synonyms of Pompia. Pompia appeared to be equally distinct from citron, lemon and sour orange based on the overall analysis of the fruit, leaf and seed phenotype, and juice chemical composition. At the leaf level, the Pompia essential oil (EO) composition is close to that of citron whereas the zest is much closer to that of sour orange., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lignification, which occurs in many horticultural fruit and vegetables, brings about undesirable texture and unfavorable consumer preference. However, this problem has rarely been studied. In this work, three pumelo cultivars cvs "Hongroumiyou" (HR), "Bairoumiyou" (BR), and "Huangroumiyou" (HuR) were stored at 25 °C for 90 days, and juice sacs were sampled to explore the lignin accumulation and its relationship to sucrose and energy depletion were investigated. The results displayed that HuR contained lower sucrose content, lower ATP level, but higher lignin content compared to BR and HR during postharvest storage, indicating that the sequence according to storage resistance on the basis of lignin content is as follows: HuR < BR < HR. Furthermore, sucrose degradation attributed to enhanced activities of neutral invertase (NI), soluble acid invertase (S-AI), cell wall-bound invertase (B-AI), and energy deficit on account of declined ATP level, showed significantly negative correlation with lignin accumulation, suggesting that lignin accumulation occurrence could induce sucrose degradation and energy deficit during postharvest storage. Additionally, higher activities of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD) could accelerate lignin synthesis and resulted in lignin accumulation during postharvest pumelo storage.

The phytochemicals in the peel of six oranges and ten mandarins including seven wild varieties and three cultivars were systematically characterised using UHPLC-Q-TOF-MS, and the correlation analysis was performed between phytochemicals and antioxidant capacity in order to investigate the phytochemical contributors to antioxidant capacity. The gradient elution was completed within 16 min and 92 compounds were undoubtedly or tentatively identified. Furthermore, the antioxidant capacities were determined using ABTS, DPPH and FRAP methods. The number of compounds, their contents and the antioxidant capacities were sequenced in the same order of the wild mandarins > cultivated mandarins > oranges. The correlation analysis that showed five compounds were significantly correlated with the antioxidant capacity and can act as main contributors to the citrus varieties with high antioxidant capacities. This study is systematic for the metabolites identification of mandarins and oranges and provides valuable information for effective utilisation of citrus peel and their bioactive compounds.

The antimicrobial activities of lemon oil based nanoemulsion and two different concentrations of lemon essential oil (100% and 10%) on food-brne pathogens (Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis and Salmonella Paratyphi A) and fish spoilage bacteria (Photobacterium damselae, Enterococcus faecalis, Vibrio vulnificus, Proteus mirabilis, Serratia liquefaciens, and Pseudomonas luteola) were compared in terms of disc diffusion, minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC). The constitutes of extracted lemon essential oil were identified by using GC-MS. Viscosity, the mean droplet size, thermodynamic stability and refractive index of nanoemulsions were determined. The main components detected in the lemon essential oil were d-limonene, p-cymene, β-pinene with percentages of 52.85%, 14.36%, and 13.69%, respectively. It was found that lemon nanoemulsion was more effective on food-borne pathogens except K. pneumoniae than 100% lemon essential oil. 10% lemon essential oil showed the highest inhibition effect on S. Paratyphi A. The conversion of the essential oil into nanoemulsion improved antimicrobial activity. According to value of MIC, both nanoemulsion and 100% essential oil inhibited bacterial growth of all of the pathogen bacteria tested whereas they were less effective on inhibition of fish spoilage bacteria. However, 10% essential oil was more effective on spoilage bacteria than pathogens. MBC showed that nanoemulsion and 100% lemon essential oil presented a noticeable bactericidal activity against S. paratyphi A whereas 10% lemon essential oil was found as ≥25 mg/mL against pathogens and spoilage bacteria. Therefore, the use of nanoemulsion based on lemon essential oil can have potential as a natural antimicrobial agent against food-borne pathogen and spoilage bacteria for fish processing industry., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In order to identify the source of Citrus grandis and evaluate its quality originate from two areas comprehensively,DNA barcode was used to identify 26 samples of C. grandis. The content of naringin,rhoifolin,naringenin and apigenin was determined by UPLC method,and the color difference was numerically studied by color difference analyzer,which was related to the effective components of C. grandis. The results showed that samples was the source of C. grandis in both regions. The ITS2 sequence length was about400-500 bp,and the sequence similarity reached 99. 82%. There was only one base deletion in the two groups. There was one base A in some medicinal materials of Guangdong at 330 bp,but no base in Chongqing. The contents of naringin and rhoifolin in Chongqing samples were higher than those in Guangdong samples,and there were statistical differences between naringenin and apigenin. The chroma value showed that L*value of Guangdong was larger,a*value was smaller,L*value of Chongqing was smaller,and a*value was larger,while the b*value of both was not significantly different; The results of correlation analysis showed that naringin,rhoifolin,naringenin were positively correlated with L*,b*value,negatively correlated with a*value,and apigenin had no correlation with L*,a*,b*value. In this study,the scientific identification and evaluation of C. grandis was carried out to provide a new idea for the further study of the rapid identification and evaluation of C. grandis.

Phenotypic characteristics, as well as the relation between carotenoid accumulation and gene expression during ripening were determined in fruits of five new hybrids between tangor cv. 'Murcott' and sweet orange cv. 'Pêra'. The genotypes were classified into the orange-like group, showing mainly epoxycarotenoids, oval fruit shape and yellowish color, or in the mandarin-like group, showing mainly β-cryptoxanthin, flattened shape and deep-orange coloration; although some hybrids presented intermediate characteristics. The diversity in carotenoid composition of hybrids and genitors were mostly explained by patterns of gene expression. High carotenoid (250-426 μg/g dry weight [dw]) and β-cryptoxanthin (81-125 μg/g dw) contents, observed in the mandarin-like group, were generally associated with high expression of upstream genes (GGPPS1, PSY, PDS). On the other hand, low expression/repression of these genes and high expression of downstream genes (BCHX and ZEP) were associated with low carotenoid (~158 μg/g dw) and β-cryptoxanthin (5-22 μg/g dw) contents and epoxycarotenoid accumulation, as occurred in the orange-like group. Breeding experiments resulted in hybrids with outstanding higher carotenoid contents than both genitors (up to 426 μg/g dw versus 158-250 μg/g dw in genitors), which was attributed to transgressive segregation. Differences among genotypes have great impact on commercial fruit quality and potential health benefits, such as the provitamin A content., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Green mold, caused by Penicillium digitatum, is the most destructive post-harvest disease in citrus. Secondary metabolites produced by fungal phytopathogens have been associated with toxicity to their respective host through the interaction with a wide range of cell targets. Natural products have also been described as important molecules for biocontrol and competition in their respective environment. For P. digitatum, the production of indole alkaloids, tryptoquialanines A and B, have been reported. However, their biological role remains unknown. Mass Spectrometry Imaging (MSI) technique was applied here for the first time to monitor the secondary metabolites produced on the orange surface during infection in order to gain insights about the P. digitatum-citrus interaction mechanisms. Through the combination of MSI and molecular networking it was possible to report, for the first time, the production of tryptoquivalines and fumiquinazolines by P. digitatum and also the accumulation of tryptoquialanines on the fruit surface from 4 to 7 d post inoculation. P. digitatum was also evaluated concerning the ability to sinthesize indole alkaloids in vivo in the different citrus hosts. The biological role of tryptoquialanines was investigated and tryptoquialanine A was submitted to insecticidal bioassays that revealed its high toxicity against Aedes Aegypti, suggesting an important insecticidal action during orange decay., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The non-destructive on-tree measurement of the chemical quality attributes of fruits belonging to the Citrus genus using rapid spectral sensors is of vital interest to citrus growers, allowing them to carry out a selective harvest of any species of Citrus fruit. With this objective, the viability of using of a handheld portable near infrared spectroscopy (NIRS) instrument to predict soluble solid content (SSC), pH, titratable acidity (TA), maturity index and BrimA, in order to measure the optimum harvest time in a group made up of 608 samples belonging to the Citrus genus (378 oranges and 230 mandarins) was evaluated. For each of the parameters analysed, both non-linear regression (LOCAL algorithm) and linear regression (Modified Partial Least Squares, MPLS) strategies were designed and compared. The use of the LOCAL algorithm in the sample group of oranges and mandarins for all the parameters analysed allowed to obtain more robust models than those obtained with MPLS regression, and it could also be extended more easily when routinely applied. The results confirm that NIRS technology combined with non-linear regression strategies such as the LOCAL algorithm can indeed respond to the needs of the Citrus growers and help them to set the optimum harvest time, in this case of oranges and mandarins, by predicting the chemical quality parameters in situ., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Long intergenic non-coding RNAs (lincRNAs) play important roles in various biological processes in plants. However, little information is known about the evolutionary characteristics of lincRNAs among closely related plant species. Here, we present a large-scale comparative study of lincRNA transcription patterns in nine citrus species. By strand-specific RNA-sequencing, we identified 18 075 lincRNAs (14 575 lincRNA loci) from 34 tissue samples. The results indicated that the evolution of lincRNA transcription is more rapid than that of mRNAs. In total, 82.8-97.6% of sweet orange (Citrus sinensis) lincRNA genes were shown to have homologous sequences in other citrus genomes. However, only 15.5-28.8% of these genes had transcribed homologous lincRNAs in these citrus species, presenting a strong contrast to the high conservation of mRNA transcription (81.6-84.7%). Moreover, primitive and modern citrus lincRNAs were preferentially expressed in reproductive and vegetative organs, respectively. Evolutionarily conserved lincRNAs showed higher expression levels and lower tissue specificity than species-specific lincRNAs. Notably, we observed a similar tissue expression pattern of homologous lincRNAs in sweet orange and pummelo (Citrus grandis), suggesting that these lincRNAs may be functionally conserved and selectively maintained. We also identified and validated a lincRNA with the highest expression in fruit that acts as an endogenous target mimic (eTM) of csi-miR166c, and two lincRNAs that act as a precursor and target of csi-miR166c, respectively. These lincRNAs together with csi-miR166c could form an eTM166-miR166c-targeted lincRNA regulatory network that possibly affects citrus fruit development., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

The traditional Chinese medicine Citri Reticulatae Pericarpium (CRP) was mainly originated from the dried pericarp of Citrus reticulata 'Chachi' (Crc), Citrus reticulata 'Dahongpao' (Crd), Citrus reticulata 'Unshiu' (Cru) and Citrus reticulata 'Tangerina' (Crt) in China. Since these four cultivars have great similarities in morphology, reliable methods to differentiate CRP cultivars have rarely been reported. To discriminate the differences of these CRP cultivars, herein an efficient and reliable method by combining metabolomics, DNA barcoding and electronic nose was first established. The hierarchical three-step filtering metabolomics analysis based on liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) indicated that 9 species-specific chemical markers including 6 flavanone glycosides and 3 polymethoxyflavones could be considered as marker metabolites for discrimination of the geoherb Crc from other cultivars. A total of 19 single nucleotide polymorphism (SNP) sites were found in nuclear internal transcribed spacer 2 (ITS2) of CRP, and three stable SNP sites (33, 128 and 174) in the ITS2 region can distinguish the four CRP cultivars. The electronic nose coupled with chemometrics could also be used to effectively distinguish Crc from other CRP cultivars. Therefore, our results indicated that the integrated method will be an effective strategy for discrimination of similar herbal medicines., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: Citri Reticulatae Pericarpium (CRP), comprising dried pericarps of Citrus reticulata Blanco and its cultivars, is popularly used for its great medicinal and dietary values. Generally, the pericarps from C. reticulate "Chachi" ("Guangchenpi" in Chinese, GCP) is considered to have superior qualities and merit premium price compared with CRP derived from other cultivars (collectively called "Chenpi" in Chinese, CP). Since its multiple origins and derived economic adulteration, it is significant to systematically compare the chemical profiles of different CRP varieties., Objective: The main objective of this work was to identify the chemical profiles of CRP from different varieties and find out potential chemical markers for differentiating GCP and CP., Methods: In the present study, a total of 42 CRP samples from 10 varieties (including GCP and CP) were analysed by ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS) for chemical profiling. Obtained MS/MS data were further employed in multivariate statistical methods to screen the main compounds which contributed to the characterisation and classification of CRP., Results: As a result, 73 compounds (mainly flavonoids) were identified or tentatively characterised in these CRP samples. Based on the obtained chemical profiles data, GCP and CP samples could be easily discriminated from each other by statistical analyses. Moreover, seven compounds were selected as having the most discriminating features which contributed to the classification of CRP., Conclusion: This work obtains a better understanding of the chemical profiles of different CRP varieties and provides a practical strategy for the authentication of GCP and CP., (© 2018 John Wiley & Sons, Ltd.)

Boron (B) toxicity in Citrus is a common physiological disorder leading to reductions in both productivity and quality. Studies on how Citrus roots evade B toxicity may provide new insight into plant tolerance to B toxicity. Here, using Illumina sequencing, differentially expressed microRNAs (miRNAs) were identified in B toxicity-treated Citrus sinensis (tolerant) and C. grandis (intolerant) roots. The results showed that 37 miRNAs in C. grandis and 11 miRNAs in C. sinensis were differentially expressed when exposed to B toxicity. Among them, miR319, miR171, and miR396g-5p were confirmed via 5'-RACE and qRT-PCR to target a myeloblastosis (MYB) transcription factor gene, a SCARECROW-like protein gene, and a cation transporting ATPase gene, respectively. Maintenance of SCARECROW expression in B treated Citrus roots might fulfill stem cell maintenance, quiescent center, and endodermis specification, thus allowing regular root elongation under B-toxic stress. Down-regulation of MYB due to up-regulation of miR319 in B toxicity-treated C. grandis roots might decrease the number of root tips, thereby dramatically changing root system architecture. Our findings suggested that miR319 and miR171 play a pivotal role in Citrus adaptation to long-term B toxicity by targeting MYB and SCARECROW , respectively, both of which are responsible for root growth and development.

Pompia is a Sardinian citrus ecotype whose botanical classification is still being debated. In the present study, the composition of Pompia peel essential oil (EO) is reported for the first time, along with that of the leaf EO, as a phytochemical contribution to the classification of this ecotype. The peel EO was tested for its antioxidant ability (with both the 2,2-diphenyl-1-picarylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays). Moreover, its antimicrobial activities were tested for the first time on dermatophytes ( Microsporum canis , Microsporum gypseum , and Trichophyton mentagrophytes ), on potentially toxigenic fungi ( Fusarium solani , Aspergillus flavus , and Aspergillus niger ) as well on bacteria ( Escherichia coli , Staphylococcus aureus , and Staphylococcus pseudointermedius ). The dominant abundance of limonene in the peel EO seems to distinguish Pompia from the Citrus spp. to which it had previously been associated. It lacks γ-terpinene, relevant in Citrus medica EO. Its relative content of α- and β-pinene is lower than 0.5%, in contrast to Citrus limon peel EO. Pompia peel and leaf EOs did not show significant amounts of linalool and linalyl acetate, which are typically found in Citrus aurantium . Pompia peel EO antioxidant activity was weak, possibly because of its lack of γ-terpinene. Moreover, it did not exert any antimicrobial effects either towards the tested bacteria strains, or to dermatophytes and environmental fungi.

Background: Clementine mandarin (Citrus clementina Hort. ex Tan.) is one of the most famous and widely grown citrus cultivars worldwide. Variations in relation to the composition and distribution of repetitive DNA sequences that dominate greatly in eukaryote genomes are considered to be species-, genome-, or even chromosome-specific. Repetitive DNA-based fluorescence in situ hybridization (FISH) is a powerful tool for molecular cytogenetic study. However, to date few studies have involved in the repetitive elements and cytogenetic karyotype of Clementine., Results: A graph-based similarity sequence read clustering methodology was performed to analyze the repetitive DNA families in the Clementine genome. The bioinformatics analysis showed that repetitive DNAs constitute 41.95% of the Clementine genome, and the majority of repetitive elements are retrotransposons and satellite DNAs. Sequential multicolor FISH using a probe mix that contained CL17, four satellite DNAs, two rDNAs and an oligonucleotide of (TTTAGGG) 3 was performed with Clementine somatic metaphase chromosomes. An integrated karyotype of Clementine was established based on unequivocal and reproducible chromosome discriminations. The distribution patterns of these probes in several Citrus, Poncirus and Fortunella species were summarized through extensive FISH analyses. Polymorphism and heterozygosity were commonly observed in the three genera. Some asymmetrical FISH loci in Clementine were in agreement with its hybrid origin., Conclusions: The composition and abundance of repetitive elements in the Clementine genome were reanalyzed. Multicolor FISH-based karyotyping provided direct visual proof of the heterozygous nature of Clementine chromosomes with conspicuous asymmetrical FISH hybridization signals. We detected some similar and variable distribution patterns of repetitive DNAs in Citrus, Poncirus, and Fortunella, which revealed notable conservation among these genera, as well as obvious polymorphism and heterozygosity, indicating the potential utility of these repetitive element markers for the study of taxonomic, phylogenetic and evolutionary relationships in the future.

Carotenoids are important antioxidant components in the human diet. To develop carotenoid-rich agricultural products by genetic intervention, understanding the genetic basis of carotenoids variation is essential. In this study, we constructed a high-density integrated genetic map with 3817 molecular markers using specific locus amplified fragment (SLAF) sequencing from a C. reticulata × P. trifoliata F 1 pseudotestcross population. A total of 17 significant quantitative trait loci (QTLs) distributed on Chromosomes (Chr) 2, 3, 5, 6, and 9 were detected to determine the carotenoid variation in the population. In particular, three QTL colocalizations for multiple carotenoid constituents were observed on Chr 2, 3, and 9, one of which was located on Chr2:34,654,608-35430715 accounted for 20.1-25.4% of the variation of luteoxanthin, auroxanthin, lutein, violaxanthin, and total carotenoid content. Overall, this study provides a genetic foundation for marker-assisted selection (MAS) breeding of nutritionally enhanced citrus fruit.

The original plant species of Fructus Aurantii are multitudinous and complex, and their requirements to the growing environment is strict. In order to clarify the original plant species and geographical distribution of Fructus Aurantii which recorded in the standards and circulated, used in commodity. The national and local standards of Chinese medicinal materials were collected and the original plants of Fructus Aurantii recoded in standards were found. Ten original plant species of Fructus Aurantii (including varieties of cultivars, the same below) were recorded in the Chinese pharmacopoeia and six local standards of Zhejiang, Yunnan and Guizhou etc. The producing areas and commodity in markets of Fructus Aurantii were investigated. The growth environment and geographical distribution of them were analyzed. There are six types of Fructus Aurantii i.e., Fructus Aurantii Chuan, Fructus Aurantii Xiang, Fructus Aurantii Jiang, Fructus Aurantii Qu, Fructus Aurantii Su, Fructus Aurantii Wen, and nineteen species of original plants in the practical commodities. There are four major Fructus Aurantii producing areas: Sichuan Basin, Dongting Lake Plain, Poyang Lake Plain, Jinqu Basin and its surrounding hilly areas. All of them are located in the area of the east longitude 104° to 121° and the northern latitudes 27° to 31°. There is a certain difference between the actual commodity and the standards of medicinal materials. It is suggested that the traditional mainstream types of Fructus Aurantii with fine quality should be accepted into Chinese Pharmacopoeia, and the types with poor quality should be withdrawn from Chinese Pharmacopoeia., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

The metabolite profile responsible for the quality of mandarin fruit is influenced by preharvest factors including genotype, rootstock, grove location, etc. In this paper, mandarin varieties were discriminated using metabolomics. Additionally, effects on metabolic profiles due to grove location and rootstock differences were also investigated. Results revealed that mandarin varieties could be differentiated using the metabolite profile, while the compositions of flavonoids have the potential for variety differentiation. With regard to fruits of the same variety, grove location might determine the overall profile of metabolites, whereas rootstock possibly affected composition of secondary metabolites. Pathway enrichment analysis demonstrated that biosynthesis pathways of terpenoids and steroids involving limonene and linalool were highly influenced by variety diversity. Moreover, the flavonoid biosynthesis pathway, involving hesperetin, naringenin, eriodictyol, and taxifolin, was indicated to have a close relationship with rootstock differentiation. This study provides useful and important information with depth for breeding and optimizing preharvest practices.

Mandarin (Citrus reticulata) is one of the most important citrus crops worldwide. Its domestication is believed to have occurred in South China, which has been one of the centers of mandarin cultivation for four millennia. We collected natural wild populations of mandarin around the Nanling region and cultivated landraces in the vicinity. We found that the citric acid level was dramatically reduced in cultivated mandarins. To understand genetic basis of mandarin domestication, we de novo assembled a draft genome of wild mandarin and analyzed a set of 104 citrus genomes. We found that the Mangshan mandarin is a primitive type and that two independent domestication events have occurred, resulting in two groups of cultivated mandarins (MD1 and MD2) in the North and South Nanling Mountains, respectively. Two bottlenecks and two expansions of effective population size were identified for the MD1 group of cultivated mandarins. However, in the MD2 group there was a long and continuous decrease in the population size. MD1 and MD2 mandarins showed different patterns of interspecific introgression from cultivated pummelo species. We identified a region of high divergence in an aconitate hydratase (ACO) gene involved in the regulation of citrate content, which was possibly under selection during the domestication of mandarin. This study provides concrete genetic evidence for the geographical origin of extant wild mandarin populations and sheds light on the domestication and evolutionary history of mandarin., (Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.)

Background: Citrus trees are among the most cultivated plants in the world, with a high economic impact. The wide sexual compatibility among relatives gave rise to a large number of hybrids that are difficult to discriminate. This work sought to explore the ability of infrared spectroscopy to discriminate among Citrus species and/or hybrids and to contribute to the elucidation of its relatedness., Results: Adult leaves of 18 distinct Citrus plants were included in this work. Near- and mid-infrared (NIR and FTIR) spectra were acquired from leaves after harvesting and a drying period of 1 month. Spectra were modelled by principal component analysis and partial least squares discriminant analysis. Both techniques revealed a high discrimination potential (78.5-95.9%), being the best results achieved with NIR spectroscopy and air-dried leaves (95.9%)., Conclusion: Infrared spectroscopy was able to successfully discriminate several Citrus species and/or hybrids. Our results contributed also to enhance insights regarding the studied Citrus species and/or hybrids. Despite the benefit of including additional samples, the results herein obtained clearly pointed infrared spectroscopy as a reliable technique for Citrus species and/or hybrid discrimination. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Citri reticulatae pericarpium (CRP), the dried pericarps of Citrus reticulata Blanco and its cultivars, has been widely used in drugs and foods in China for centuries. In this study, an accurate and feasible analytical method based on HS-SPME-GC-MS coupled with multivariate statistical analyses was developed to comprehensively compare volatile compounds of pericarps derived from Citrus reticulata "Chachi" ("Guangchenpi" in Chinese, GCP) and other cultivars of Citrus reticulata Blanco ("Chenpi" in Chinese, CP). Principal component analysis, hierarchical cluster analysis, and orthogonal partial least-squares-discrimination analysis were performed to extract meaningful attributes from volatile profiles based on GC-MS data. Results indicated that samples from GCP and CP could easily be differentiated, and seven potential chemical markers were screened for the quality control of CRP. This study illuminated the volatile profile in CRP, and provides a practical method for the authentication of CRP varieties.

The citrus industry is facing an unprecedented challenge from Huanglongbing (HLB). All cultivars can be affected by the HLB-associated bacterium 'Candidatus Liberibacter asiaticus' (CLas) and there is no known resistance. Insight into HLB pathogenesis is urgently needed in order to develop effective management strategies. Here, we use Sec-delivered effector 1 (SDE1), which is conserved in all CLas isolates, as a molecular probe to understand CLas virulence. We show that SDE1 directly interacts with citrus papain-like cysteine proteases (PLCPs) and inhibits protease activity. PLCPs are defense-inducible and exhibit increased protein accumulation in CLas-infected trees, suggesting a role in citrus defense responses. We analyzed PLCP activity in field samples, revealing specific members that increase in abundance but remain unchanged in activity during infection. SDE1-expressing transgenic citrus also exhibit reduced PLCP activity. These data demonstrate that SDE1 inhibits citrus PLCPs, which are immune-related proteases that enhance defense responses in plants.

Key Message: Host perception of Phytophthora nicotianae switching to necrotrophy is fundamental for disease tolerance of citrus. It involves an HR-like response, strengthening of the cell wall structure and hormonal signaling. Stem rot caused by P. nicotianae is a worldwide disease of several important crops, including citrus. Given the growing awareness of chemical fungicides drawbacks, genetic improvement of citrus rootstocks remains the best alternative. However, the molecular basis underlying the successful response of resistant and/or tolerant genotypes remains poorly understood. Therefore, we performed a transcriptomic analysis to examine the differential defense response to P. nicotianae of two germplasms-tolerant sour orange (SO, Citrus aurantium) and susceptible Madam Vinous (MV, C. sinensis)-in both the biotrophic and necrotrophic phases of host-pathogen interaction. Our results revealed the necrotrophic phase as a decisive turning point, since it included stronger modulation of a number of genes implicated in pathogen perception, signal transduction, HR-like response, transcriptional reprogramming, hormone signaling, and cell wall modifications. In particular, the pathogen perception category reflected the ability of SO to perceive the pathogen even after its switch to necrotrophy, and thus to cope successfully with the infection, while MV failed. The concomitant changes in genes involved in the remaining functional categories seemed to prevent pathogen spread. This investigation provided further understanding of the successful defense mechanisms of C. aurantium against P. nicotianae, which might be exploited in post-genomic strategies to develop resistant Citrus genotypes.

The inherent allergens of citrus fruits, such as Cit s 1, Cit s 2, Cit s 3 can cause allergic reactions. A better understanding of the genetic factors (cultivar to cultivar) affecting the allergenic potential of citrus fruits would be beneficial for further identification of hypoallergenic genotypes. In the present study, an immunoblotting quantification approach was adopted to assess the potential allergenicity of 21 citrus cultivars, including nine subgroups (tangerine, satsuma, orange, pummelo, grapefruit, lemon, kumquat, tangor, and tangelo). To prepare highly sensitive and specific rabbit polyclonal antibodies, antigenicity of purified rCit s 1.01, rCit s 2.01, and rCit s 3.01 peptides were enhanced with high epitope density in a single protein molecule. The data integration of three citrus allergen quantifications demonstrated that the four pummelo cultivars (Kao Phuang Pummelo, Wanbai Pummelo, Shatian Pummelo, and Guanxi Pummelo) were potential hypoallergenic, compared with other 8 subgroups. Moreover, the immunological analyses with sera of allergic subjects revealed that Shatian Pummelo and Guanxi Pummelo showed the lowest immunoreactivity in 8 representative citrus cultivars. These potential hypoallergenic genotypes are of great significance to not only allergic consumers but also citrus breeders in the genetic improvement of hypoallergenic citrus as breeding resources.