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Citrus unshiu Marc. cv. Miyagawa-wase is one of the most widely cultivated citrus varieties on Jeju Island in Republic of Korea. Mutation breeding is a useful tool for inducing genetic diversity by causing genomic mutations in a short period of time. We previously conducted mutation breeding using gamma irradiation to develop new varieties of C. unshiu. Here, we describe one of these varieties, Yein-early, which has a redder peel, greater hardness, and higher sugar content compared with the wild type (WT). Yein-early leaves also showed a unique phenotype compared with the WT, characterized by longer longitudinal length, shorter transverse length, stronger curling, and longer petiole length. Genome resequencing of Yein-early and the WT uncovered significant single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels). These variations were crucial in identifying molecular markers unique to Yein-early. In addition, we developed an allele-specific PCR marker specifically targeting a homozygous SNP in Yein-early that distinguishes it from the WT and other citrus varieties. This study contributes to the understanding of pigment synthesis in fruits and provides a valuable tool for selection of the novel Yein-early variety in citrus breeding programs.

Vascular dementia (VaD) is the second most common type of dementia after Alzheimer’s disease. In our previous studies, we showed that wheat bran extract (WBE) reduced white matter damage in a rat VaD model and improved memory in a human clinical trial. However, starch gelatinization made the large-scale preparation of WBE difficult. To simplify the manufacturing process and increase efficacy, we attempted to find a decoction containing an optimum ratio of wheat bran, sliced citrus peel, and sliced jujube (WCJ). To find an optimal ratio, the cell survival of C6 (rat glioma) cultured under hypoxic conditions (1% O2) was measured, and apoptosis was assessed. To confirm the efficacies of the optimized WCJ for VaD, pupillary light reflex, white matter damage, and the activation of astrocytes and microglia were assessed in a rat model of bilateral common carotid artery occlusion (BCCAO) causing chronic hypoperfusion. Using a combination of both searching the literature and cell survival experiments, we chose 6:2:1 as the optimal ratio of wheat bran to sliced citrus peel to sliced jujube to prepare WCJ. We showed that phytic acid contained only in wheat bran can be used as an indicator component for the quality control of WCJ. We observed in vitro that the WCJ treatment improved cell survival by reducing apoptosis through an increase in the Bcl-2/Bax ratio. In the BCCAO experiments, the WCJ-supplemented diet prevented astrocytic and microglial activation, mitigated myelin damage in the corpus callosum and optic tract, and, consequently, improved pupillary light reflex at dosages over 100 mg/kg/day. The results suggest that the consumption of WCJ can prevent VaD by reducing white matter damage, and WCJ can be developed as a safe, herbal medicine to prevent VaD.

Citrus species are widely cultivated across the globe and frequently encounter drought stress during their growth and development phases. Previous research has indicated that citrus species synthesize flavonoids as a response mechanism to drought stress. This study aimed to comprehensively quantify and analyze the presence of 85 distinct flavonoids in the leaf and root tissues of lemon (drought susceptible) and sour orange (drought tolerant). In drought-stressed sour orange roots, flavonoids, such as isosakuranin, mangiferin, trilobatin, liquiritigenin, avicularin, silibinin, and glabridin, were more elevated than control sour orange roots and drought-stressed lemon roots. Additionally, hydroxysafflor yellow A, cynaroside, tiliroside, and apigenin 7-glucoside were increased in drought-stressed sour orange leaves compared to drought-stressed lemon leaves. Under drought stress, flavonoids such as (-)-epigallocatechin, silibinin, benzylideneacetophenone, trilobatin, isorhamnetin, 3,7,4′-trihydroxyflavone, and liquiritigenin were significantly increased, by 3.01-, 3.01-, 2.59-, 2.43-, 2.07-, 2.05-, and 2.01-fold, in sour orange roots compared to control sour orange roots. Moreover, the total flavonoid content and antioxidant capacity were significantly increased in drought-stressed sour orange leaves and root tissues compared to drought-stressed lemon leaves and root tissues. The expression levels of genes involved in flavonoid biosynthesis were highly expressed in sour orange leaves and roots, compared to lemon leaves and root tissues, post-drought stress. These findings indicate that lemons fail to synthesize protective flavonoids under drought conditions, whereas sour orange leaves and root tissues enhance flavonoid synthesis, with higher antioxidant activities to mitigate the adverse effects of reactive oxygen species generated during drought stress.

The symptoms of huanglongbing (HLB), a disease caused by the bacterium Candidatus Liberibacter asiaticus (CLas), are visible on the canopy of citrus plants. They include mottling of leaves followed by dropping and lopsided fruits with premature dropping. Loss in phloem functionality and degradation of the root system are also HLB symptoms with a severe impact on plant growth and production. Some Citrus relatives, such as Poncirus trifoliata and its hybrids, have shown more tolerance to HLB disease and low titers of CLas compared to Citrus species, but little is known about the effects of CLas on their root system. In this study, we investigated the effects of CLas-infected ‘Valencia’ scion on the citrandarin IAC3222 (a hybrid between P. trifoliata and Sunki mandarin) used as rootstock as well as interstock between ‘Valencia’ and Swingle citrumelo rootstock. At 13 months post-inoculation, the cycle threshold values (CT) for CLas in the infected scion samples indicated a high CLas titer (from 15.9 to 22.7) regardless of the rootstock variety or interstock used. However, no CLas-positive samples were detected in the roots of IAC3222 (CT ranging from 37.9 to 40.0), in contrast to all Swingle roots (CT ranging from 27.9 to 31.3). Both root volume and mass were reduced in IAC3222 compared to uninfected ‘Valencia’ scion, suggesting that scion infection damages roots, regardless of whether they are contaminated or not by CLas. The damage to the root system of IAC3222 was significantly less severe than that of the Swingle rootstock. Multivariate hierarchical analysis considering all evaluated parameters clustered the CLas-infected plants grafted on IAC3222 together with the non-inoculated plants. We concluded that the IAC3222 rootstock was less affected by the CLas-infected scion compared to the Swingle rootstock and is a promising rootstock to minimize the HLB effect on plant development.

Lemon fruit (Citrus limon (L.) Burm.) is highly appreciated by consumers due to its antioxidant properties and health benefits. However, its shelf life can be limited by various factors, reducing the economy, and thereafter, new strategies to maintain the quality of lemons are necessary. Melatonin is a derivative of tryptamine, which is ubiquitously found in plants and has a wide range of functions regulating numerous physiological processes in plants. During two consecutive harvests, we evaluated the effect of preharvest treatments with melatonin on crop yield and on quality and functional properties of fruit of lemon cv. Verna at harvest and weekly after storage up to 28 days at 2 and 10 °C plus 2 days at 20 °C. Melatonin was applied as foliar spray treatments at dosages of 0.1, 0.3, and 0.5 mM and at three different stages of fruit development. The results showed that melatonin treatment had a positive impact on crop yield as well as in fruit quality parameters, such as firmness, content of bioactive compounds, and antioxidant activity, especially for a 0.5 mM dose. Taking all these effects into account, the application of melatonin along the growth cycle of fruit development could be considered a non-contaminant and eco-friendly tool for improving crop yield and quality of ‘Verna’ lemons at harvest and during postharvest storage.

Transposable elements (TEs) significantly contribute to the evolution and diversity of plant genomes. In this study, we explored the roles of TEs in the genomes of Citrus and Citrus-related genera by constructing a pan-genome TE library from 20 published genomes of Citrus and Citrus-related accessions. Our results revealed an increase in TE content and the number of TE types compared to the original annotations, as well as a decrease in the content of unclassified TEs. The average length of TEs per assembly was approximately 194.23 Mb, representing 41.76% (Murraya paniculata) to 64.76% (Citrus gilletiana) of the genomes, with a mean value of 56.95%. A significant positive correlation was found between genome size and both the number of TE types and TE content. Consistent with the difference in mean whole-genome size (39.83 Mb) between Citrus and Citrus-related genera, Citrus genomes contained an average of 34.36 Mb more TE sequences than Citrus-related genomes. Analysis of the estimated insertion time and half-life of long terminal repeat retrotransposons (LTR-RTs) suggested that TE removal was not the primary factor contributing to the differences among genomes. These findings collectively indicate that TEs are the primary determinants of genome size and play a major role in shaping genome structures. Principal coordinate analysis (PCoA) of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) identifiers revealed that the fragmented TEs were predominantly derived from ancestral genomes, while intact TEs were crucial in the recent evolutionary diversification of Citrus. Moreover, the presence or absence of intact TEs near the AdhE superfamily was closely associated with the bitterness trait in the Citrus species. Overall, this study enhances TE annotation in Citrus and Citrus-related genomes and provides valuable data for future genetic breeding and agronomic trait research in Citrus.

Understanding canopy nitrogen (N) and phosphorus (P) differences is crucial for optimizing plant nutrient distribution and management. This study evaluated leaf N and P content in citrus trees across three cultivation modes: traditional mode (TM), wide-row and narrow-plant mode (WRNPM), and fenced mode (FM). We used hyperspectral data for non-destructive quantification and compared 1080 leaf samples from upper, middle, and lower canopy layers. Four models—Random Forest (RF), Backpropagation Neural Network (BPNN), Partial Least Squares (PLS), and Support Vector Machine (SVM)—were employed for leaf N and P estimation. Results showed that the TM had significantly lower N content compared to the WRNPM and FM, while the WRNPM exhibited higher P content. The canopy layer had minimal impact on N and P in the FM, and leaves in the upper layer had higher nutrient content in the WRNPM and TM. RF provided the best estimation accuracy, with R2 values of 0.66 for N and 0.72 for P. The cultivation mode and canopy layer significantly influenced the estimation accuracy, with the TM yielding the highest R2, followed by the WRNPM and FM obtaining the lowest accuracy. The labor-saving cultivation mode had different nutrient utilization efficiency compared to the TM. The cultivation mode and canopy layer should be considered when hyperspectral data were used for estimating the leaf N and P content. The study can offer new insights for precise fertilization strategies in fruit trees.

Plant endophytic microbial communities consist of many latent plant pathogens and, also, many pathogen-related species with reduced virulence. Though with a long history of co-evolution, the diversity and composition of the endophytic mycobiota, especially the pathogen-related fungal groups, has been under-investigated in Citrus (C.). Based on the amplicon sequencing of fungal internal transcribed spacer (ITS), the leaf endophytic mycobiota were profiled on citrus varieties from different citrus-producing regions. The pomelo variety shared significantly distinctive leaf mycobiota when compared to the mandarin and sweet orange; these conform to their host genetic relationships. In addition, a data set of 241 citrus-related fungi, including 171 (71%) pathogens and potential pathogens, was summarized from previous studies. Under the criteria of local BLAST (covered ITS nucleotide ≥ 150 bp, sequence identity ≥ 99%), a total of 935 fungal operational taxonomic units (OTUs) were assigned to 62 pathogen-related fungal groups, representing 14.9% of the relative abundance in the whole community. Of which, the top groups consisted of Colletotrichum gloeosporioides (mean relative abundance, 4.3%), Co. citricola and Co. karstii (2.7%), Zasmidium citri-griseum (2.4%), and Z. fructigenum (1.4%). At the genus level, the ratio of the pathogen-related fungal groups in 64% of fungal genera (16 out of 25) exceeded 50%, which are the solely or mainly occurring fungi of their genus in citrus. Our study suggests that the leaf endophytic compartment may be an important place for the growth of latent pathogens.

The prolonged utilization of copper (Cu)-containing fungicides results in Cu accumulation and affects soil ecological health. Thus, a pot experiment was conducted using Citrus reticulata cv. Shatangju with five Cu levels (38, 108, 178, 318, and 388 mg kg−1) to evaluate the impacts of the soil microbial processes, chemistry properties, and citrus growth. These results revealed that, with the soil Cu levels increased, the soil total Cu (TCu), available Cu (ACu), organic matter (SOM), available potassium (AK), and pH increased while the soil available phosphorus (AP) and alkali-hydrolyzable nitrogen (AN) decreased. Moreover, the soil extracellular enzyme activities related to C and P metabolism decreased while the enzymes related to N metabolism increased, and the expression of soil genes involved in C, N, and P cycling was regulated. Moreover, it was observed that tolerant microorganisms (e.g., p_Proteobacteria, p_Actinobacteria, g_Lysobacter, g_Sphingobium, f_Aspergillaceae, and g_Penicillium) were enriched but sensitive taxa (p_Myxococcota) were suppressed in the citrus rhizosphere. The citrus biomass was mainly positively correlated with soil AN and AP; plant N and P were mainly positively correlated with soil AP, AN, and acid phosphatase (ACP); and plant K was mainly negatively related with soil β−glucosidase (βG) and positively related with the soil fungal Shannon index. The dominant bacterial taxa p_Actinobacteriota presented positively correlated with the plant biomass and plant N, P, and K and was negatively correlated with plant Cu. The dominant fungal taxa p_Ascomycota was positively related to plant Cu but negatively with the plant biomass and plant N, P, and K. Notably, arbuscular mycorrhizal fungi (p_Glomeromycota) were positively related with plant P below soil Cu 108 mg kg−1, and pathogenic fungi (p_Mortierellomycota) was negatively correlated with plant K above soil Cu 178 mg kg−1. These findings provided a new perspective on soil microbes and chemistry properties and the healthy development of the citrus industry at increasing soil Cu levels.

Agriculture faces the dual challenge of increasing food production and safeguarding the environment. Climate change exacerbates this challenge, reducing crop yield and biomass due to drought stress, especially in semi-arid regions where Citrus plants are cultivated. Understanding the molecular mechanisms underlying drought tolerance in Citrus is crucial for developing adaptive strategies. Plants of two citrus rootstocks, Carrizo Citrange and Bitters (C22), were grown in aerated half-strength Hoagland’s nutrient solution. Post-acclimation, the plants were exposed to a solution containing 0% (control) or 15% PEG-8000 for 10 days. Leaf malonyl dialdehyde (MDA) and hydrogen peroxide (H2O2) content were measured to assess the reached oxidative stress level. Total RNA was extracted, sequenced, and de novo-assembled. Weighted Gene Correlation Network Analysis (WGCNA) was conducted to examine the relationship between gene expression patterns and the levels of MDA and H2O2 used as oxidative stress indicators. Plant visual inspection and MDA and H2O2 contents clearly indicate that Bitters is more tolerant than Carrizo towards PEG-induced drought stress. RNA-Seq analysis revealed a significantly higher number of differentially expressed genes (DEGs) in Carrizo (6092) than in Bitters (320), with most being associated with drought sensing, ROS scavenging, osmolyte biosynthesis, and cell wall metabolism. Moreover, the WGCNA identified transcription factors significantly correlated with MDA and H2O2 levels, thus providing insights into drought-coping strategies and offering candidate genes for enhancing citrus drought tolerance.

Oxygen heterocyclic compounds play a beneficial role in plants, and their presence in foods, such as Citrus fruits, cinnamon, carrots, and parsley, has been documented in recent years. Published research articles reported several extractions and chromatographic techniques for their determination. The aim of this review was to take into consideration the research articles published from 2016 to 2024 in which the authors developed extraction and chromatographic analysis methods of oxygen heterocyclic compounds in foods. The objective of this review was to assist researchers in choosing the best approach for their future work by identifying all the possible approaches to characterize coumarins, furocoumarins, and polymethoxyflavones in foodstuffs.

Facility-forcing cultivation could effectively improve the quality of ‘Beni Madonna’ citrus (Citrus nankao × C. amakusa) and advance the ripening period. However, segment drying (KS) before harvest caused fruit quality deterioration and commodity value loss. In this research, we investigated the physiological and molecular characteristics involved in citrus segment drying under facility-forcing cultivation. The juice yield, sugar, acid, vitamin C, and lignin contents in KS fruits were significantly decreased, and the contents of pectin and cellulose were significantly increased. The relative contents of abscisic acid and abscisic acid glucosyl ester in KS fruits were significantly decreased. A total of 1215 differentially expressed genes (DEGs) were screened by transcriptome sequencing. DEGs were significantly enriched in water metabolism, sugar metabolism, transportation, cell wall and phenylpropanoid biosynthesis, and plant hormone signal transduction. The decrease in water absorption and sugar synthesis, the increase of pectin and cellulose synthesis, and the decrease in ABA accumulation may be the main reasons that cause citrus fruit segments to dry under facility-forcing cultivation.

In the framework of our studies on Citrus cultivars in Greece, the chemical composition of the essential oils (EOs) from the peels and leaves of orange, pummelo, and tangelo (mandarin × grapefruit hybrid) cultivated in Greece have been studied. All EOs have been analyzed through GC-MS, and a total of 47 and 87 metabolites were identified in the peels and leaves, respectively. These metabolites are classified into the chemical groups of terpenes, alcohols, aldehydes, esters, ketones, and organic acids. Limonene was the most abundant compound in the peel EOs. Moreover, bioactive polymethoxyflavones (PMFs) were isolated and structurally determined from the peels of orange and tangelo, highlighting them as a good potential source of natural PMFs. All EOs were evaluated for their antimicrobial activity against nine human pathogenic microorganisms (six bacteria and three fungi), showing an interesting profile. The EOs from the peels of all Citrus species exhibited a stronger antimicrobial activity compared to those from the leaves. The susceptibility of the assayed Gram-positive bacteria was observed to be greater than that of Gram-negative bacteria, while the fungi were also relatively less resistant than bacteria. The rootstock choice did not influence the EO profile of the fruit peel but exerted an influence on the chemical profile of the leaves.

Citrus cultivars have various temperature needs for development and output at different times from flowering to harvesting, making climate one of the numerous factors that affect citrus’ productivity and quality. In this study, the yield and heat unit requirements for several commercial citrus species over several seasons from 2010/2011 to 2021/2022 in Egypt were investigated. For this investigation, the time of flowering and the time of fruit harvesting were recorded. However, the required heat units from flowering to fruit harvesting were calculated based on daily records of air temperature, taking into account that all results below zero (negative results) are not used and all maximum air temperatures above 35.0 °C (≥35.1) are changed to 35.0 °C. In addition, the base air temperature of 13.0 °C was utilized for determining the required heat units. The results showed that in the experimental area, the overall mean of minimum air temperature, maximum air temperature, air relative humidity, and precipitation during the experimental periods had values of 15.2 °C, 28.70 °C, 59.3%, and 0.3 mm/day, respectively. Moreover, the lowest number of days required from flowering to fruit harvesting was observed to be 290.0 days for Fremont mandarin. Furthermore, the highest number of days required from flowering to fruit harvesting was observed to be 482 and 440 days, respectively, for Bearss Seedless lime and Valencia orange. Our study has highlighted a notable diversity among the investigated citrus cultivars, particularly highlighting specific cultivars that possess high yield. The cultivar that shows the greatest yield over the investigated seasons of the study was Valencia orange with 147.6 kg/tree. Moreover, the average values of the heat unit requirements for Washington Navel orange, Valencia orange, Murcott mandarin, Fremont mandarin, and Bearss Seedless lime were 3112.7, 3628.3, 3221.9, 3027.6, and 4398.4 °C day, respectively. This is the first report regarding the heat unit’s determination for several citrus cultivars grown in Egypt, and we expect this research will provide a new awareness in accepting and discovering novel locations where citrus cultivars can be positively developed in Egypt. It will also prove to be a source of basic information for the development of the citrus crop model.

Potassium is an essential mineral nutrient for citrus growth and stress response. In this study, the HAK/KUP/KT gene family was identified from the genome of trifoliate orange (Poncirus trifoliata). The physical and chemical properties, chromosomal location, gene structure, evolutionary relationship, conserved motifs, and tissue expression characteristics were analyzed. The expression characteristics under low potassium and salt stress were analyzed by fluorescence quantitative PCR. The function of PtKUP10 was investigated by heterologous expression in Arabidopsis thaliana. The results showed that at least 18 PtKUPs were distributed in seven chromosomes. Phylogenetic analysis showed that four PtKUPs clustered in clade I, which mediated the high-affinity potassium absorption. Gene expression analysis showed that four PtKUPs were highly expressed in root, seven PtKUPs were up-regulated by low potassium stress, and nine PtKUPs were up-regulated by salt stress. The cis-acting elements on the promoter of PtKUPs were predominantly involved in stress and hormone responses. Overexpression of PtKUP10 in Arabidopsis thaliana could enhance salt tolerance by accumulating more potassium in the shoot and reducing sodium content in the shoots and roots. These results indicated that PtKUPs play important roles in potassium absorption and salt stress response, and PtKUP10 might enhance salt tolerance by maintaining potassium and sodium homeostasis.

The flavonoids in citrus fruits are crucial physiological regulators and natural bioactive products of high pharmaceutical value. Melatonin is a pleiotropic hormone that can regulate plant morphogenesis and stress resistance and alter the accumulation of flavonoids in these processes. However, the direct effect of melatonin on citrus flavonoids remains unclear. In this study, nontargeted metabolomics and transcriptomics were utilized to reveal how exogenous melatonin affects flavonoid biosynthesis in “Bingtangcheng” citrus fruits. The melatonin treatment at 0.1 mmol L−1 significantly increased the contents of seven polymethoxylated flavones (PMFs) and up-regulated a series of flavonoid pathway genes, including 4CL (4-coumaroyl CoA ligase), FNS (flavone synthase), and FHs (flavonoid hydroxylases). Meanwhile, CHS (chalcone synthase) was down-regulated, causing a decrease in the content of most flavonoid glycosides. Pearson correlation analysis obtained 21 transcription factors co-expressed with differentially accumulated flavonoids, among which the AP2/EREBP members were the most numerous. Additionally, circadian rhythm and photosynthesis pathways were enriched in the DEG (differentially expressed gene) analysis, suggesting that melatonin might also mediate changes in the flavonoid biosynthesis pathway by affecting the fruit’s circadian rhythm. These results provide valuable information for further exploration of the molecular mechanisms through which melatonin regulates citrus fruit metabolism.

Arbuscular mycorrhizal (AM) fungi can enhance the uptake of soil nutrients and water by citrus, promoting its growth. However, the specific mechanisms underlying the action of AM fungi in promoting the growth of citrus were not fully elucidated. This study aimed to explore the role of AM fungi Funneliformis mosseae in the regulatory mechanisms of P. trifoliata growth. Pot experiments combined with non-targeted metabolomics methods were used to observe the growth process and changes in metabolic products of P. trifoliata under the conditions of F. mosseae inoculation. The results showed that F. mosseae could form an excellent symbiotic relationship with P. trifoliata, thereby enhancing the utilization of soil nutrients and significantly promoting its growth. Compared with the control, the plant height, stem diameter, number of leaves, and aboveground and underground dry weight in the F. mosseae inoculation significantly increased by 2.57, 1.29, 1.57, 4.25, and 2.78 times, respectively. Moreover, the root system results confirmed that F. mosseae could substantially promote the growth of P. trifoliata. Meanwhile, the metabolomics data indicated that 361 differential metabolites and 56 metabolic pathways were identified in the roots of P. trifoliata and were inoculated with F. mosseae. This study revealed that the inoculated F. mosseae could participate in ABC transporters by upregulating their participation, glycerophospholipid metabolism, aminoacyl tRNA biosynthesis, tryptophan metabolism and metabolites from five metabolic pathways of benzoxazinoid biosynthesis [mainly enriched in lipid (39.50%) and amino acid-related metabolic pathways] to promote the growth of P. trifoliata.

For perennial plants, newly emerged organs are fresh hot spots for environmental microbes to occupy and assemble to form mature microbial communities. In the microbial community, some commensal fungi can play important roles in microbial succession, thus significantly improving host plant growth and disease resistance. However, their participating patterns in microbial assembly and succession remain largely unknown. In this study, we profiled the fungal community and found a similar fungal succession pattern of spring-emerged leaves from March to October in two pomelo orchards. Specifically, the fungal species, tracked on the old leaves, dominated the spring leaves after emergence and then decreased in relative abundance. This reduction in priority effects on the spring leaves was then followed by an increase in the number of observed species, Shannon and phylogenetic diversity indices, and the pathogen-associated fungal groups. In addition, we found that the temporal fungal succession on the spring leaves highly correlated with the disease occurrence in the orchards and with the temperature and precipitation variation from spring to summer. Of the pathogen-associated fungal groups, an increase in the relative abundance of Mycosphaerellaceae, hosting the causal agent of citrus greasy spot, correlated with the occurrence of the disease, while the relative abundance of Diaporthaceae, hosting the causal agent of melanose, was extremely low during the fungal succession. These results confirm that the two kinds of pathogen-associated fungal groups share different lifestyles on citrus, and also suggest that the study of temporal fungal succession in microbial communities can add to our understanding of the epidemiology of potential plant pathogens.

To address the current issues of complex structures and low accuracies in citrus pest identification models, a lightweight pest identification model was proposed. First, a parameterized linear rectification function was introduced to avoid neuronal death. Second, the model’s attention to pest characteristics was improved by incorporating an improved mixed attention mechanism. Subsequently, the network structure of the original model was adjusted to reduce architectural complexity. Finally, by employing transfer learning, an SCHNet model was developed. The experimental results indicated that the proposed model achieved an accuracy rate of 94.48% with a compact size of 3.84 MB. Compared to the original ShuffleNet V2 network, the SCHNet model showed a 3.12% accuracy improvement while reducing the model size by 22.7%. The SCHNet model exhibited an excellent classification performance for citrus pest identification, enabling the accurate identification of citrus pests.

Huanglongbing (HLB) is a severe citrus disease worldwide. Wild Australian limes like Citrus australasica, C. inodora, and C. glauca possess beneficial HLB resistance traits. Individual trees of the three taxa were extensively used in a breeding program for over a decade to introgress resistance traits into commercial-quality citrus germplasm. We generated high-quality, phased, de novo genome assemblies of the three Australian limes using PacBio long-read sequencing. The genome assembly sizes of the primary and alternate haplotypes were determined for C. australasica (337 Mb/335 Mb), C. inodora (304 Mb/299 Mb), and C. glauca (376 Mb/379 Mb). The nine chromosome-scale scaffolds included 86–91% of the genome sequences generated. The integrity and completeness of the assembled genomes were estimated to be at 97.2–98.8%. Gene annotation studies identified 25,461 genes in C. australasica, 27,665 in C. inodora, and 30,067 in C. glauca. Genes belonging to 118 orthogroups were specific to Australian lime genomes compared to other citrus genomes analyzed. Significantly fewer canonical resistance (R) genes were found in C. inodora and C. glauca (319 and 449, respectively) compared to C. australasica (576), C. clementina (579), and C. sinensis (651). Similar patterns were observed for other gene families associated with potential HLB resistance, including Phloem protein 2 (PP2) and Callose synthase (CalS) genes predicted in the Australian lime genomes. The genomic information on Australian limes developed in the present study will help understand the genetic basis of HLB resistance.

Evaptotranspiration (ETc) is a crucial link in the farmland water cycle process. To accurately obtain the citrus ETc in different slope positions, the METRIC, RSEB, and FAO Penman–Monteith (P-M) models were constructed based on unmanned aerial vehicle (UAV) multispectral images to invert the ETc values. The ETc of citrus calculated by the P-M model was used as a reference standard, and the accuracy of the ETc inversion was evaluated by the METRIC model and the RSEB model. The results showed that the R2, RMSE, and SE of the METRIC model and the RSEB model were 0.396 and 0.486, 4.940 and 3.010, and 4.570 and 2.090, respectively, indicating a higher accuracy of the RSEB model for inverting the ETc values. Furthermore, the accuracy of the RSEB model could be improved by introducing the optimal correction coefficient (after correction: RMSE = 1.470, SE = 0.003). Based on the modified RSEB model, the ETc values of the citrus in different slope positions were obtained. We also found that the middle slope ETc > the top slope ETc > the bottom slope ETc, indicating that the slope position indeed affected the citrus ETc. This research provides a favorable framework for the ETc inversion, and the results are of theoretical and practical importance to realize crop water conservation.

Citrus (Citrus reticulata) is one of the world’s most widely planted and highest-yielding fruit trees. Citrus fruits are rich in a variety of nutrients. The content of citric acid plays a decisive role in the flavor quality of the fruit. There is a high organic acid content in early-maturing and extra-precocious citrus varieties. Reducing the amount of organic acid after fruit ripening is significant to the citrus industry. In this study, we selected a low-acid variety, “DF4”, and a high-acid variety, “WZ”, as research materials. Through WGCNA analysis, two differentially expressed genes, citrate synthase (CS) and ATP citrate-pro-S-lyase (ACL), were screened out, which related to the changing citric acid. The two differentially expressed genes were preliminarily verified by constructing a virus-induced gene-silencing (VIGS) vector. The VIGS results showed that the citric acid content was negatively correlated with CS expression and positively correlated with ACL expression, while CS and ACL oppositely control citric acid and inversely regulate each other. These results provide a theoretical basis for promoting the breeding of early-maturing and low-acid citrus varieties.

Citrus orchards in semi-arid regions are increasingly exposed to drought conditions due to climate change. This study compared the physiological and growth responses of ‘W. Murcott’ tangor (WM) grafted onto Citrus macrophylla (M), Swingle citrumelo (SC), C-35 citrange (C35), or bitter citrandarin (C22) rootstock subjected to two irrigation treatments: daily irrigation to replace 100% of the water lost daily by evapotranspiration (ET; control treatment) or daily irrigation to replace 75% of the water lost daily by ET (water deficit treatment). For trees in each treatment, leaf gas exchange, relative chlorophyll content, chlorophyll fluorescence, midday stem water potential, trunk cross-sectional area, and shoot length were measured 46 days after treatments were initiated. The results showed that WM on SC or C22 rootstock exhibited isohydric behavior, where decreased stomatal conductance limited transpiration in the water deficit treatment. WM on M rootstock exhibited an anisohydric response in the water deficit treatment, where there was no stomatal control of water loss by transpiration. Among the rootstocks tested for WM, the most tolerant to soil water deficit was SC, whereas trees on M rootstock were the most negatively affected by soil water deficit.

Florida ranks among the most important citrus growing regions in the USA. The present study investigates the occurrence, diversity, and pathogenicity of fungal species associated with symptomatic sweet orange (Citrus sinensis) cv. Valencia plants and fruit. The survey was conducted on twigs and fruit collected in Southwest Florida during 2022. Based on morphological and molecular characteristics, the identified isolates belonged to the species Lasiodiplodia iraniensis, Diaporthe pseudomangiferae, and Diaporthe ueckerae. The pathogenicity of representative isolates was evaluated on citrus fruit and plants. Lasiodiplodia iraniensis was the most virulent on fruit and plants, followed by Diaporthe pseudomangiferae. Diaporthe ueckerae had the lowest virulence on fruit, and it was not pathogenic to plants. In vitro tests were performed to assess the effect of temperature on mycelial radial growth. The optimum temperature of growth ranged from 26.0 to 28.4 °C for all the evaluated species, and L. iraniensis showed the fastest mycelial growth. This study represents the first report of L. iraniensis as a causal agent of tree dieback and fruit stem-end rot on C. sinensis worldwide. Moreover, D. pseudomangiferae and D. ueckerae are reported here for the first time in association with citrus diseases worldwide.

Arbuscular mycorrhizal fungi (AMF) are known to enhance plant growth via stimulation of root system development. However, the extent of their effects and underlying mechanisms across different citrus genotypes remain to be fully elucidated. This study investigates the impact of Funneliformis mosseae (F. mosseae) inoculation on plant growth performance, root morphology, phosphorus (P), and indole-3-acetic acid (IAA) concentrations, as well as the expression of related synthesis and transporter genes in three citrus genotypes: red tangerine (Citrus tangerine ex. Tanaka), kumquat (Fortunella margarita L. Swingle), and fragrant citrus (Citrus junos Sieb. ex. Tanaka). Following 12 weeks of inoculation, significant improvements were observed in plant height, shoot and root biomass, total root length, average root diameter, second-order lateral root development, root hair density, and root hair length across all genotypes. Additionally, F. mosseae inoculation significantly increased root P and IAA concentrations in the three citrus genotypes. Notably, phosphatase activity was enhanced in F. margarita but reduced in C. tangerine and C. junos following inoculation. Gene expression analysis revealed a universal upregulation of the P transporter gene PT5, whereas expressions of the auxin synthesis gene YUC2, transporter gene LAX2, and phosphatase gene PAP1 were commonly downregulated. Specific to genotypes, expressions of YUC5, LAX5, PIN2, PIN3, PIN6, and expansin genes EXPA2 and EXPA4 were significantly upregulated in C. tangerine but downregulated in F. margarita and C. junos. Principal component analysis and correlation assessments highlighted a strong positive association between P concentration, P and auxin synthesis, and transporter gene expressions with most root morphology traits, except for root average diameter. Conversely, IAA content and phosphatase activities were negatively correlated with these root traits. These findings suggest that F. mosseae colonization notably enhances plant growth and root system architecture in citrus genotypes via modifications in P transport and IAA accumulation, indicating a complex interplay between mycorrhizal symbiosis and host plant physiology.

Seedless is a highly valued commercial characteristic in the citrus industry, both for fresh consumption and for processed markets. In this study, the ‘Succari Sweet Orange’ (WT) and its seedless mutant ‘Juxiangyuan’ (MT), which originated from a bud mutation, were selected to study the formation of a citrus seedless phenotype. Microscopic analysis of MT’s floral organs, including anther and ovary cross-sections, provides insights into its seedless phenotype compared to the original seeded cultivar. Additionally, pollen features, viability, and in vitro germination were examined to determine the cause of seedlessness. MT exhibited significant developmental deformities in both male and female gametes, with pollen grain analysis indicating a high rate of deformity (41.48%), low viability (9.59%), and minimal in vitro germination (9.56%). Hybridization experiments were conducted to assess male and female sterility and pollen incompatibility. Both WT and MT exhibited parthenocarpic development. Notably, MT fruit produced with an average of 3.51 seeds pollinated to WT, despite severe pollen abortion of MT. MT, however, produced seedless fruit through self-breeding or cross-breeding with other varieties, demonstrating stable female sterility. Despite reduced pollen quantity and viability in the seedless mutant ‘Juxiangyuan’, its seedlessness primarily stems from female sterility. This study contributed to a deeper understanding of seedless formation in ‘Juxiangyuan’ and provided valuable information relevant to its commercial cultivation.

Green mold caused by Penicillium digitatum (Pers.:Fr.) Sacc is the most prevalent postharvest rot concerning citrus fruits. Using the subtractive suppression hybridization (SSH) technique, different P. digitatum genes have been identified that could be involved in virulence during citrus infection in the early stages, a crucial moment that determines whether the infection progresses or not. To this end, a comparison of two P. digitatum strains with high and low virulence has been carried out. We conducted a study on the gene expression profile of the most relevant genes. The results indicate the importance of transcription and regulation processes as well as enzymes involved in the degradation of the plant cell wall. The most represented expressed sequence tag (EST) was identified as PDIP_11000, associated with the FluG domain, which is putatively involved in the activation of conidiation. It is also worth noting that PDIP_02280 encodes a pectin methyl esterase, a cell wall remodeling protein with a high expression level in the most virulent fungal strains, which is notably induced during citrus infection. Furthermore, within the group with the greatest representation and showing significant induction in the early stages of infection, regulatory proteins (PDIP_68700, PDIP_76160) and a chaperone (PDIP_38040) stand out. To a lesser extent, but not less relevant, it is worth distinguishing different regulatory proteins and transcription factors, such as PDIP_00580, PDIP_49640 and PDIP_78930.

The GLK gene family plays a crucial role in the regulation of chloroplast development and participates in chlorophyll synthesis. However, the precise mechanism by which GLK contributes to citrus’s chlorophyll synthesis remains elusive. The GLK gene family causes variations in the photosynthetic capacity and chlorophyll synthesis of different citrus varieties. In this study, we identified tissue-specific members and the key CcGLKs involved in chlorophyll synthesis. A total of thirty CcGLK transcription factors (TFs) were discovered in the citrus genome, distributed across all nine chromosomes. The low occurrence of gene tandem duplication events and intronic variability suggests that intronic variation may be the primary mode of evolution for CcGLK TFs. Tissue-specific expression patterns were observed for various GLK family members; for instance, CcGLK12 and CcGLK15 were specifically expressed in the skin, while CcGLK30 was specific to the ovary, and CcGLK10, CcGLK6, CcGLK21, CcGLK2, CcGLK18, CcGLK9, CcGLK28, and CcGLK8 were specifically expressed in the leaves. CcGLK4, CcGLK5, CcGLK11, CcGLK23, CcGLKl7, CcGLK26, and CcGLK20 may participate in the regulation of the ALA, prochlorophylate, protoporphyrin IX, Mg-protoporphyrin IX, Chl b, T-Chl, MG-ProtoIX ME, and POR contents in citrus.

Desalinated seawater (DSW) has emerged as a promising solution for irrigation in regions facing water scarcity. However, adopting DSW may impact the existing cultivation model, given the presence of potentially harmful elements, among other factors. A three-year experiment was carried out to assess the short-term effects of four irrigation waters—freshwater (FW), DSW, a mix 1:1 of FW and DSW (MW), and DSW with low boron (B) concentration (DSW–B)—on a ‘Rio Red’ grapefruit orchard. These irrigation waters exhibited varying levels of phytotoxic elements, some potentially harmful to citrus trees. Sodium (Na+) and chloride (Cl−) concentrations exceeded citrus thresholds in all treatments, except in DSW−B, whilst B exceeded toxicity levels in DSW and MW treatments. Leaf concentrations of Cl− and Na+ remained low in all treatments, whereas B approached toxic levels only in DSW and MW–irrigated trees. The rapid growth of the trees, preventing excessive accumulation through a dilution effect, protected the plants from significant impacts on nutrition and physiology, such as gas exchange and chlorophyll levels, due to phytotoxic elements accumulation. Minor reductions in photosynthesis in DSW–irrigated trees were attributed to high B in leaves, since Cl− and Na+ remained below toxic levels. The accelerated tree growth effectively prevented the substantial accumulation of phytotoxic elements, thereby limiting adverse effects on tree development and yield. When the maturation of trees reaches maximal growth, the potential accumulation of phytotoxic elements is expected to increase, potentially influencing tree behavior differently. Further study until the trees reach maturity is imperative for comprehensive understanding of the long-term effects of desalinated seawater irrigation.

Citrus fruits are economically significant crops worldwide, as they contain various bioactive compounds that possess health-promoting properties. Carotenoids, as the most important component in citrus, exhibit notable pharmacological activities, such as antioxidation and anticancer, which make carotenoids valuable in the prevention and treatment of breast cancer. In this study, after treatment with carotenoid extracts from XiYou (XY) and ZaoHongQiCheng (ZH), we evaluated the cytotoxicity, apoptosis, antioxidant system, and oxidative stress induced by ROS overproduction and MMP damage in MDA-MB-231 cells. The analysis confirmed that cell proliferation was inhibited in a concentration-dependent manner, accompanied by G0/G1 arrest and cell apoptosis. XY and ZH promoted the accumulation of ROS, decreased MMP, increased malondialdehyde (MDA) levels, consumed glutathione (GSH), and reduced the activity of antioxidant enzymes (peroxidase (POD), catalase (CAT), glutathione reductase (GR), and superoxide dismutase (SOD)). Meanwhile, XY and ZH induced apoptosis through the mitochondrial pathway by significantly upregulated P53, BAX, caspase-3, caspase-7, and caspase-9 gene expression levels and downregulated Bcl-2. Carotenoid-rich extracts were found to cause oxidative stress by enhancing ROS production through their pro-oxidative potential, and the aggravation of oxidative processes promotes apoptosis in MDA-MB-231 cells. These results indicate that citrus carotenoids can be used as potential pro-oxidants and have the potential to be developed into products for the prevention or treatment of breast cancer.

Rapid advancements in technologies provide various tools to analyze fruit crop genomes to better understand genetic diversity and relationships and aid in breeding. Genome-wide single nucleotide polymorphism (SNP) genotyping arrays offer highly multiplexed assays at a relatively low cost per data point. We report the development and validation of 1.4M SNP Axiom® Citrus HD Genotyping Array (Citrus 15AX 1 and Citrus 15AX 2) and 58K SNP Axiom® Citrus Genotyping Arrays for Citrus and close relatives. SNPs represented were chosen from a citrus variant discovery panel consisting of 41 diverse whole-genome re-sequenced accessions of Citrus and close relatives, including eight progenitor citrus species. SNPs chosen mainly target putative genic regions of the genome and are accurately called in both Citrus and its closely related genera while providing good coverage of the nuclear and chloroplast genomes. Reproducibility of the arrays was nearly 100%, with a large majority of the SNPs classified as the most stringent class of markers, “PolyHighResolution” (PHR) polymorphisms. Concordance between SNP calls in sequence data and array data average 98%. Phylogenies generated with array data were similar to those with comparable sequence data and little affected by 3 to 5% genotyping error. Both arrays are publicly available.

The citrus blackfly (CBF), Aleurocanthus woglumi Ashby, is an exotic pest native to Southeast Asia that has spread rapidly to the world’s main centers of citrus production, having been recently introduced to Brazil. In this study, a maximum entropy niche model (MaxEnt) was used to predict the potential worldwide distribution of CBF under current and future climate change scenarios for 2030 and 2050. These future scenarios came from the Coupled Model Intercomparison Project Phase 6 (CMIP6), SSP1-2.6, and SSP5-8.5. The MaxEnt model predicted the potential distribution of CBF with area under receiver operator curve (AUC) values of 0.953 and 0.930 in the initial and final models, respectively. The average temperature of the coldest quarter months, precipitation of the rainiest month, isothermality, and precipitation of the driest month were the strongest predictors of CBF distribution, with contributions of 36.7%, 14.7%, 13.2%, and 10.2%, respectively. The model based on the current time conditions predicted that suitable areas for the potential occurrence of CBF, including countries such as Brazil, China, the European Union, the USA, Egypt, Turkey, and Morocco, are located in tropical and subtropical regions. Models from SSP1-2.6 (2030 and 2050) and SSP5-8.5 (2030) predicted that suitable habitats for CBF are increasing dramatically worldwide under future climate change scenarios, particularly in areas located in the southern US, southern Europe, North Africa, South China, and part of Australia. On the other hand, the SSP5-8.5 model of 2050 indicated a great retraction of the areas suitable for CBF located in the tropical region, with an emphasis on countries such as Brazil, Colombia, Venezuela, and India. In general, the CMIP6 models predicted greater risks of invasion and dissemination of CBF until 2030 and 2050 in the southern regions of the USA, European Union, and China, which are some of the world’s largest orange producers. Knowledge of the current situation and future propagation paths of the pest serve as tools to improve the strategic government policies employed in CBF’s regulation, commercialization, inspection, combat, and phytosanitary management.

Citrus is an important type of fruit, with antioxidant bioactivity. However, the variations in the antioxidant ability of different tissues in citrus and its metabolic and molecular basis remain unclear. Here, we assessed the antioxidant capacities of 12 tissues from Citrus reticulata ‘Ponkan’, finding that young leaves and root exhibited the strongest antioxidant capacity. Secondary metabolites accumulated differentially in parts of the citrus plant, of which flavonoids were enriched in stem, leaf, and flavedo; phenolic acids were enriched in the albedo, while coumarins were enriched in the root, potentially explaining the higher antioxidant capacities of these tissues. The spatially specific accumulation of metabolites was related to the expression levels of biosynthesis-related genes such as chalcone synthase (CHS), chalcone isomerase (CHI), flavone synthase (FNS), O-methyltransferase (OMT), flavonoid-3′-hydroxylase (F3′H), flavonoid-6/8-hydroxylase (F6/8H), p-coumaroyl CoA 2′-hydroxylase (C2′H), and prenyltransferase (PT), among others, in the phenylpropane pathway. Weighted gene co-expression network analysis (WGCNA) identified modules associated with flavonoids and coumarin content, among which we identified an OMT involved in coumarin O-methylation, and related transcription factors were predicted. Our study identifies key genes and metabolites influencing the antioxidant capacity of citrus, which could contribute to the enhanced understanding and utilization of bioactive citrus components.

Citrus plants are prone to phosphorus (P) deficiency, especially in acidic soil, making them more dependent on root-associated endophytic fungi for growth and development. Beni-Madonna, a hybrid of Citrus nanko × C. amakusa, is a citrus known as “tangor” that is highly popular in China and other parts of the world due to its deep red color and jelly-like flesh. In this study, the inoculation response to two arbuscular mycorrhizal fungi (Diversispora versiformis, Dv; Funneliformis mosseae, Fm) and an endophytic fungus (Serendipita indica, Si) with regard to the growth, leaf gas exchange, light energy efficiency, P levels, acid phosphatase activity, and expression of the purple acid phosphatase (PAP) and phosphate transporter (PT) genes through a potted experiment using Beni-Madonna (tangor) citrus plants grafted on trifoliate orange (Poncirus trifoliata) was studied. Two years following inoculation, the root fungal colonization rates of inoculated plants were significantly increased, accompanied by an increase in plant height and stem diameter, with Si presenting the best effect. Fungal inoculations also significantly increased the leaf chlorophyll index, nitrogen-balance index, photosynthesis rate, stomatal conductance, transpiration rate, photosynthetic efficiency, and quantum yield of PSII, while it reduced photoinhibition and heat dissipation, with Si having the best effect on light energy efficiency. Si significantly increased leaf and root P levels, as well as root CsPAP1‒3, CsPT2, CsPT3, and CsPT7 expression and soil acid phosphatase activity; Fm in mycorrhizal fungi significantly increased leaf and root P levels, as well as root CsPT1 and CsPT7 expression and root/soil acid phosphatase activity. These observations suggested an advantage of Si over the other two AMF in terms of improved plant growth and P acquisition, while Fm displayed prominent effects on increased photosynthetic efficiency.

Bergaptol (5-hydroxypsoralen or 5-hydroxyfuranocoumarin) is a naturally occurring furanocoumarin widely found in citrus fruits, which has multiple health benefits. Nonetheless, no specific review articles on bergaptol have been published. Compiling updated information on bergaptol is crucial in guiding future research direction and application. The present review focuses on the research evidence related to the pharmacological properties and toxicity of bergaptol. Bergaptol has anti-inflammatory, antioxidant, anti-cancer, anti-osteoporosis, anti-microbial, and anti-lipidemic effects. It can inhibit the activities of cytochrome P450s (CYP), especially CYP2C9 and CYP3A4, thereby affecting the metabolism and concentrations of some drugs and toxins. Compared with other coumarins, bergaptol has the least potency to inhibit CYP3A4 in cancer cells. Instead, it can suppress drug efflux transporters, such as P-glycoprotein, thereby overcoming chemotherapeutic drug resistance. Furthermore, bergaptol has antimicrobial effects with a high potential for inhibition of quorum sensing. In vivo, bergaptol can be retained in plasma for longer than other coumarins. Nevertheless, its toxicity has not been clearly reported. In vitro study suggests that, unlike most furocoumarins, bergaptol is not phototoxic or photomutagenic. Existing research on bergaptol has mostly been conducted in vitro. Further in vivo and clinical studies are warranted to identify the safe and effective doses of bergaptol for its multimodal application.

Adapting agricultural systems to face persistent environmental hazards is at the center of global concerns. In line with this, understanding and highlighting the structural characteristics of agroforestry systems could strengthen their resilience in terms of disease management. This study was conducted to evaluate the effect of shade on the intensity of citrus leaf and fruit spot disease caused by Pseudocercospora (PLFSD). Investigations to assess the effects of shade components on the incidence of PLFSD were carried out on 15-year-old tangerine trees in a cocoa-based agroforestry plot (Bokito) during four fruits seasons. Tangerines under the shade of large forest trees were compared to others located under full sunlight. A complementary experiment was conducted on young grapefruit plants in an orchard with mango and avocado groves in Foumbot. Three shading conditions, i.e., under avocado trees, under mango trees, and without shade, were explored. Data on shade and PLFSD incidence were collected and analyzed. Our findings show that PLFSD incidence was null on tangerine leaves from trees under shade compared to those under full sunlight. The same trends were observed in fruits under shade and under full sunlight. Disease incidence on grapefruit leaves was lower on trees under shade compared to those under full sunlight. In short, shade trees appear to constitute potential physical barriers to disease progression. This study also highlights disease spatial distribution as beyond 12 m of distance between neighboring trees, no spatial dependence of disease spread was observed. Management actions based on the distance between citrus trees and regulating shade are envisaged.

Citrus fruits stand as pivotal and extensively cultivated fruit crops on a global scale, boasting substantial economic and nutritional significance. Despite their paramount importance, citrus growers and the industry face a formidable obstacle in the form of post-harvest losses caused by plant pathogens. Effectively addressing this challenge has become imperative. The predominant approach to tackle these pathogens has traditionally involved the use of chemical fungicides. However, the escalating environmental concerns associated with chemical interventions, coupled with a growing consumer preference for pesticide-free produce, have catalyzed an earnest quest for alternative methods of disease control in the citrus industry. The antagonistic yeasts hold great promise as biocontrol agents for mitigating post-harvest fungal diseases in citrus. In this regard, this review summarizes the current state of knowledge regarding the study of yeast strains with biocontrol potential. Thus, the various modes of action employed by these yeasts and their effectiveness against prominent citrus pathogens such as Penicillium digitatum, Penicillium italicum and Geotrichum citri were discussed. Additionally, the review delved into the challenges associated with the practical implementation of yeast-based biocontrol strategies in citrus post-harvest management and investigated the potential of yeast-based approaches to enhance the safety and quality of citrus produce, while reducing the reliance on chemical fungicides and contributing to the sustainable and environmentally responsible future of the citrus industry.

The volatiles of different aerial parts of three kumquat species (Fortunella margarita Swingle–Nagami, Fortunella japonica Swingle–Marumi, and Fortunella crassifolia Swingle–Meiwa) growing in Greece were analyzed via GC-MS and evaluated for their antimicrobial properties against nine human pathogenic microorganisms. A total number of 23 compounds were identified in the peel, 38 in the leaves, and 30 in the flowers of the examined species. Limonene was the dominant metabolite in the peels of all three species, germacrene-D was present in the leaves of Nagami and Marumi kumquats, while limonene was the most abundant in the flower of Marumi and Meiwa kumquat but with significant differences in the composition of the total fracture of the essential oil, since compounds with high antimicrobial activity were only present in the flower of Meiwa kumquat. The essential oils from the leaf and peel of the three kumquat species were either inactive or showed weak antimicrobial activity, respectively, against Gram-positive and Gram-negative bacterial strains and pathogenic fungi. Only the essential oil from the flower of F. crassifolia Swingle (Meiwa) showed a stronger effect (MIC values 3.5–7.48 mg/mL) against all the assayed microorganisms. Furthermore, through multivariate statistical analysis, we studied the relationships between the samples regarding their origin (species and plant part), as well as between the chemical composition of the corresponding essential oils and their antimicrobial activity. Considering its chemical profile and antimicrobial activity, the Greek Meiwa flowers’ essential oil seemed a promising essential oil for further exploitation in the food and/or medicinal industry.

In this work, the peels of red and blonde oranges as well as lemons were efficiently (5.75–9.65% yield) extracted by hydroalcoholic solution with ultrasound assistance and employed as active molecule sources in the preparation of functional gummies. Antioxidant performances of the hydroalcoholic extracts were characterized by colorimetric assays, whereas LC–HRMS analyses identified the main bioactive compounds (phenolic acids and flavonoids). The highest scavenging activity was recorded for lemon extract in an aqueous environment (IC50 = 0.081 mg mL−1). An ecofriendly grafting procedure was performed to anchor polyphenols to gelatin chains, providing macromolecular systems characterized by thermal analysis and antioxidant properties. Scavenger abilities (IC50 = 0.201–0.454 mg mL−1) allowed the employment of the conjugates as functional ingredients in the preparation of gummies with remarkable antioxidant and rheological properties over time (14 days). These findings confirmed the possible employment of highly polluting wastes as valuable sources of bioactive compounds for functional gummies preparation.

‘Albedo bluing’ of fruits occurs in many varieties of citrus, resulting in a significant reduction in their commercial value. We first presented a breakthrough method for successfully extracting and purifying the ‘albedo bluing’ substance (ABS) from citrus fruits, resulting in the attainment of highly purified ABS. Then, HPLC and UPLC-QTOF-MS were used to prove that ABS in the fruits of three citrus varieties (Citrus reticulate Blanco cv. ‘Gonggan’, ‘Orah’, and ‘Mashuiju’) are identical. However, the chemical structure of ABS remains elusive for many reasons. Fortunately, a more stable derivative of ABS (ABS-D) was successfully obtained. Through various analytical techniques such as HRESIMS, 1D and 2D NMR, and chemical shift calculation, ABS-D was identified as 2,4-dihydroxy-6-(β-D-glucopyranosyloxy)phenyl(5,6-dihydroxy-7-(β-D-glucopyranosyloxy)benzo[d]thiazol-2-yl)methanone, indicating that both ABS and its derivative belong to a rare category of benzothiazole glucosides. Furthermore, both ABS and ABS-D demonstrated potent antioxidant abilities. These findings lay the groundwork for further elucidating the chemical structure of ABS and the causative mechanism of the ‘albedo bluing’ phenomenon in citrus fruits.

p-Synephrine is a common alkaloid widely distributed in citrus fruits. However, the effects of p-synephrine on the metabolic profiles of individuals with energy abnormalities are still unclear. In the study, we investigated the effect of p-synephrine on energy homeostasis and metabolic profiles using a high fat diet (HFD)-induced mouse model. We found that p-synephrine inhibited the gain in body weight, liver weight and white adipose tissues weight induced by HFD. p-Synephrine supplementation also reduced levels of serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) but not to a statistically significant degree. Histological analysis showed that HFD induced excessive lipid accumulation and glycogen loss in the liver and adipocyte enlargement in perirenal fat tissue, while p-synephrine supplementation reversed the changes induced by HFD. Moreover, HFD feeding significantly increased mRNA expression levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and reduced the mRNA expression level of interleukin-10 (IL-10) compared to the control group, while p-synephrine supplementation significantly reversed these HFD-induced changes. Liver and serum metabolomic analysis showed that p-synephrine supplementation significantly altered small molecule metabolites in liver and serum in HFD mice and that the changes were closely associated with improvement of energy homeostasis. Notably, amino acid metabolism pathways, both in liver and serum samples, were significantly enriched. Our study suggests that p-synephrine improves energy homeostasis probably by regulating amino acid metabolism in HFD mice, which provides a novel insight into the action mechanism of p-synephrine modulating energy homeostasis.

Diabetes mellitus and related metabolic and vascular impairments are notable health problems. Fruits and vegetables contain phenolics that are beneficial to metabolic and oxidative health and useful in preventing associated disease. Scientific evidence has shown that some bioactive phenolics are more abundant in the non-edible parts (especially the peels) of many fruits than in their respective edible tissues. Fruits belonging to the Citrus and Prunus genera are commonly consumed worldwide, including in South Africa, and their non-edible wastes (peel and seed) have been shown to have antioxidative, metabolic and vascular pharmacological potentials and medicinal phytochemistry. It is therefore imperative to evaluate the pharmacological actions and phytochemical properties of the non-edible wastes of these fruits and understand how they could potentially be of medicinal relevance in oxidative, metabolic and vascular diseases, including diabetes, oxidative stress, obesity, hypertension and related cardiovascular impairments. In the absence of a previous review that has concomitantly presented the medicinal potentials of fruits wastes from both genera, this review presents a critical analysis of previous and recent perspectives on the medicinal potential of the non-edible wastes from the selected Citrus and Prunus fruits in metabolic, vascular and oxidative health. This review further exposes the medicinal phytochemistry, while elucidating the underlying mechanisms through the fruit wastes potentiates their therapeutic effects. A literature search was carried out on “PubMed” to identify peer-reviewed published (mostly 2015 and beyond) studies reporting the antidiabetic, antioxidative, antihypertensive, anti-hyperlipidemic and anti-inflammatory properties of the non-edible parts of the selected fruits. The data of the selected studies were analyzed to understand the bioactive mechanisms, bioactive principles and toxicological profiles. The wastes (seed and peel) of the selected fruits had antioxidant, anti-obesogenic, antihypertensive, anti-inflammatory, antidiabetic and tissue protective potentials. Some phenolic acids and terpenes, as well as flavonoids and glycosides such as narirutin, nobiletin, hesperidin, naringin, naringenin, quercetin, rutin, diosmin, etc., were the possible bioactive principles. The peel and seed of the selected fruits belonging to the Citrus and Prunus genera are potential sources of bioactive compounds that could be of medicinal relevance for improving oxidative, metabolic and vascular health. However, there is a need for appropriate toxicological studies.

Fruit cracking affects both the yield and economic efficiency of citrus; however, the underlying mechanism remains unclear. Therefore, this study focused on resistant and susceptible cultivars to identify the mechanisms underlying fruit cracking. The results showed that in ‘Mingrijian’, pectin morphological transformation and hemicellulose and lignin degradation in the pericarp were important contributing factors. During the critical fruit-cracking period (115–150 days after flowering), the water-soluble pectin, protopectin, and lignin contents in the pericarp of ‘Daya’ presented inverse changes relative to those in ‘Mingrijian’, thus enhancing the mechanical properties and resistance of pericarp. From 115 to 150 days after flowering, the soluble sugar content in the pulp of ‘Mingrijian’ increased rapidly by 97.35%, aiding in pulp water absorption and expansion. Moreover, the soluble protein content in the pericarp of ‘Mingrijian’ exhibited a declining trend and was lower than that of ‘Daya’, thus affecting the overall metabolism. The superoxide dismutase (SOD) activity in the pericarp of ‘Mingrijian’ gradually decreased from 115 to 180 days after flowering, while the peroxidase (POD) activity remained at a low level, resulting in weaker antioxidant capacity and lower environmental resistance. This study provides valuable insights into the mechanisms of citrus fruit cracking, laying the foundation for preventive and control strategies.

Low-temperature stress is an important limiting factor affecting citrus growth and fruit yields. Therefore, increasing citrus cold stress tolerance may enhance the growth, yield, and quality of citrus production in marginal areas. The objective of this study was to determine the efficacy of silicon (Si) fertilizer application on cold-tolerance enhancement in citrus. Two citrus cultivars (Delta and Nules) were subjected to Si fertilization (control, 1000 mg L−1) and cold-stress temperature treatments (control and 0 °C for 72 h) using a 2 × 2 × 2 factorial treatment structure with six replications. Leaf gas exchange and chlorophyll fluorescence parameters, such as net photosynthetic rate (A), stomatal conductance (gs), transpiration rate (Tr), internal CO2 concentration (Ci), intrinsic water-use efficiency (iWUE), minimal fluorescence (Fo), maximum fluorescence (Fm), maximum quantum efficiency of PSII primary photochemistry of dark-adapted leaves (Fv/Fm), maximum quantum efficiency of PSII primary photochemistry of dark-/light-adapted leaves (F’v/F’m), electron transport rate (ETR), non-photochemical quenching (NPQ), and the relative measure of electron transport to oxygen molecules (ETR/A), were measured. The application of Si drenching to trees that were subsequently exposed to cold stress reduced gs, Tr, and Ci but improved iWUE and Fo in both cultivars compared to the Si-untreated trees. In addition, specific adaptation mechanisms were found in the two citrus species; NPQ and ETR were improved in Si-treated Valencia trees, while A, Fm, and ETR/A were improved in Clementine trees under chilling stress conditions. The current research findings indicate the potential of Si application to enhance cold stress tolerance in citrus, which can provide a strategy for growing citrus in arid and semi-arid regions that may experience cold stress. Overall, after the application of silicon drenching, the cold-sensitive citrus Valencia cultivar became as cold-tolerant as the cold-tolerant Clementine cultivar.

Citrus plants rely heavily on arbuscular mycorrhizal fungi (AMF) due to their lack of root hairs. Most experiments have been conducted with AMF inoculation under potted conditions, while field inoculation of AMF on citrus, especially a high economic hybrid tangor variety Beni-Madonna (Citrus nanko × C. amakusa), has been rarely recorded. This study aimed to analyze the effects of two AMF inoculations (a single Funneliformis mosseae and a mixture of F. mosseae, Diversispora versiformis, and Rhizophagus intraradices) on the internal and external fruit quality, leaf antioxidant defense system, and soil fertility and structure of top-worked Beni-Madonna tangor citrus trees. Three and a half years after AMF inoculations, soil hyphal length and root mycorrhizal colonization rate increased by 61.2–101.8% and 15.85–29.6% in inoculated plants, respectively. Inoculated trees had higher external fruit coloration value, fruit horizontal diameter, and fruit weight, and lower fruit rigidity than uninoculated trees. AMF-inoculated trees had higher glucose levels of fruit peels, fructose and sucrose levels of fruit fleshes, and the ratio of fruit soluble solids/titratable acids, as well as lower titratable acids concentrations than non-AMF-inoculated trees. AMF inoculation significantly increased leaf nitrogen balance index, chlorophyll index, peroxidase, catalase, superoxide dismutase, and glutathione reductase activities, as well as reduced glutathione and oxidized glutathione concentrations, resulting in lower hydrogen peroxide and malondialdehyde levels when compared to the uninoculated treatment. In addition, inoculated trees presented higher soil nutrient levels, including organic carbon, available K, and Olsen-P as, well as soil aggregate stability (based on mean weight diameter) than uninoculated trees. This study concluded that field AMF inoculation improved fruit quality, enhanced leaf antioxidant defense system, and improved soil fertility of Beni-Madonna trees, with mixed AMF being prominent in improving fruit quality and F. mosseae being prominent in enhancing leaf antioxidant defense system and improving soil fertility.

Candidatus Liberibacter asiaticus (CLas) is associated with Citrus Huanglongbing (HLB), a devastating disease in the US. Previously, we conducted a two-year-long monthly HLB survey by quantitative real-time PCR using root DNA fractions prepared from 112 field grapefruit trees grafted on sour orange rootstock. Approximately 10% of the trees remained CLas-free during the entire survey period. This study conducted 16S metagenomics using the time-series root DNA fractions, monthly prepared during twenty-four consecutive months, followed by microbial co-occurrence network analysis to investigate the microbial factors contributing to the CLas-free phenotype of the aforementioned trees. Based on the HLB status and the time when the trees were first diagnosed as CLas-positive during the survey, the samples were divided into four groups, Stage H (healthy), Stage I (early), II (mid), and III (late) samples. The 16S metagenomics data using Silva 16S database v132 revealed that HLB compromised the diversity of rhizosphere microbiota. At the phylum level, Actinobacteria and Proteobacteria were the predominant bacterial phyla, comprising >93% of total bacterial phyla, irrespective of HLB status. In addition, a temporal change in the rhizosphere microbe population was observed during a two-year-long survey, from which we confirmed that some bacterial families differently responded to HLB disease status. The clustering of the bacterial co-occurrence network data revealed the presence of a subnetwork composed of Streptomycetaceae and bacterial families with plant growth-promoting activity in Stage H and III samples. These data implicated that the Streptomycetaceae subnetwork may act as a functional unit against HLB.

Cold is one of the major limiting factors for citrus production, particularly extreme cold waves. Therefore, it is of great importance to develop cold-tolerant varieties and clarify their cold tolerance mechanisms in citrus breeding. In this study, comparative transcriptomic and physiological analyses were performed to dissect the cold tolerance mechanism of Guijing2501 (GJ2501), a new satsuma mandarin (Citrus unshiu) variety with about 1 °C lower LT50 (the median lethal temperature) relative to Guijing (GJ). The physiological analysis results revealed that GJ2501 is more cold-tolerant with less photoinhibition, PSII photodamage, and MDA accumulation, but higher POD activity than GJ under cold stress. Comparative transcriptomic analysis identified 4200 DEGs between GJ and GJ2501, as well as 4884 and 5580 up-regulated DEGs, and 5288 and 5862 down-regulated DEGs in response to cold stress in GJ and GJ2501, respectively. “Photosynthesis, light harvesting” and “photosystem” were the specific and most significantly enriched GO terms in GJ2501 in response to cold stress. Two CuELIP1 genes (encoding early light-induced proteins) related to the elimination of PSII photodamage and photoinhibition were remarkably up-regulated (by about 1000-fold) by cold stress in GJ2501 as indicated by RT-qPCR verification. Overexpression of CuELIP1 from GJ2501 in transgenic Arabidopsis protected PSII against photoinhibition under cold stress. Taken together, the cold tolerance of GJ2501 may be ascribed to its higher photoprotective capacity under cold stress.

Huanglongbing (HLB; also known as citrus greening) is the most destructive bacterial disease of citrus worldwide with no known sustainable cure yet. Herein, we used non-targeted metabolomics and transcriptomics to prove that γ-aminobutyric acid (GABA) accumulation might influence the homeostasis of several metabolic pathways, as well as antioxidant defense machinery, and their metabolism-related genes. Overall, 41 metabolites were detected in ‘Valencia’ sweet orange (Citrus sinensis) leaf extract including 19 proteinogenic amino acids (PAA), 10 organic acids, 5 fatty acids, and 9 other amines (four phenolic amines and three non-PAA). Exogenous GABA application increased most PAA in healthy (except L-threonine, L-glutamine, L-glutamic acid, and L-methionine) and ‘Candidatus L. asiaticus’-infected citrus plants (with no exception). Moreover, GABA accumulation significantly induced L-tryptophan, L-phenylalanine, and α-linolenic acid, the main precursors of auxins, salicylic acid (SA), and jasmonic acid (JA), respectively. Furthermore, GABA supplementation upregulated most, if not all, of amino acids, phenolic amines, phytohormone metabolism-related, and GABA shunt-associated genes in both healthy and ‘Ca. L. asiaticus’-infected leaves. Moreover, although ‘Ca. L. asiaticus’ induced the accumulation of H2O2 and O2•− and generated strong oxidative stress in infected leaves, GABA possibly stimulates the activation of a multilayered antioxidative system to neutralize the deleterious effect of reactive oxygen species (ROS) and maintain redox status within infected leaves. This complex system comprises two major components: (i) the enzymatic antioxidant defense machinery (six POXs, four SODs, and CAT) that serves as the front line in antioxidant defenses, and (ii) the non-enzymatic antioxidant defense machinery (phenolic acids and phenolic amines) that works as a second defense line against ‘Ca. L. asiaticus’-induced ROS in citrus infected leaves. Collectively, our findings suggest that GABA might be a promising alternative eco-friendly strategy that helps citrus trees battle HLB particularly, and other diseases in general.

Citrus tristeza virus (CTV) is one of the most destructive diseases affecting citrus and is a major cause of reductions in citrus yield. CTV epidemics have caused the death of millions of citrus trees globally. The present study aims to evaluate citrus cultivars against CTV and its vector (aphid) population. The highest levels of infection and vector population were recorded in Mangal Singh, whereas the lowest were found in the early fruiter (20%). Early fruiter had a maximum level of tolerance against Citrus tristeza virus. CTV is replicated in the phloem cells of plants and is transmitted by the aphid specie Aphis gossypii. Thus, the maximum vector population mirrors the highest infection. Chemical plant nutrients, including micro-mix (Zn, Fe, Cu, Mn), NPK, zinc, and the insecticide Lufenoron, were used to limit the impact of CTV and A.gossyii. Lufenuron caused maximum disease inhibition, followed by the plant nutrients zinc, NPK, and micro-mix, respectively. However, Lufenoron significantly decreased the population of Aphis gossypi. The results indicate that the early fruiter has the lowest percent disease index and vector population. Moreover, Lufenuron is the best solution for controlling vector population and disease inhibition.

In this study, we compared the fruit quality and color of ‘Kiyomi’ (WT) and its mutant (MT) grafted on Ziyang xiangcheng (Cj) (WT/Cj, MT/Cj), and the MT grafted on Trifoliate orange (Pt) (MT/Pt). The differences in sugar, organic acid, flavonoids, phenols, and volatile substances of the three materials were also analyzed by high performance liquid chromatography (HPLC) and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC–MS). The results showed significant differences in the appearance of WT/Cj, MT/Cj, and MT/Pt. MT/Pt, compared to WT/Cj, MT/Cj, had lower sugar, acid, phenol and flavonoid contents in the pulp. However, MT/Pt pulp was higher in vitamin C (VC), and the peel had significantly higher total phenol and flavonoid contents. In terms of pulp, WT/Cj had the greatest diversity of volatile organic compounds (VOCs). 4-methyl-1-pentanol was significantly higher in MT/Cj pulp, while MT/Pt pulp had a unique octanoic acid, methyl ester. VOCs were more diverse in the peels of the three materials. β-Myrcene and valencen were significantly higher in MT/Cj peels. In contrast, 16 unique VOCs were detected in MT/Pt, and D-limonene content was significantly higher than in WT/Cj and MT/Cj. The results suggest Trifoliate orange is a suitable rootstock for MT.