Citrus Hybrids Research Articles

New insights in the flavor and chemistry of Huanglongbing tolerant citrus hybrids with/without Poncirus trifoliata in their pedigree

IntroductionCitrus hybrids with Poncirus trifoliata L. Raf. introgression have gained interest due to their tolerance to Huanglongbing (HLB), a devastating disease for Florida citrus agriculture. While these hybrids inherit disease tolerance from P. trifoliata, they sometimes also suffer from undesirable off-flavors.MethodsA selection of thirteen genotypes were harvested over the 2020-2021 and 2021-2022 seasons. Their juices were evaluated by a trained sensory panel and were comprehensively analyzed for their chemical makeup, including soluble solids content (SSC), titratable acidity (TA), volatiles, flavonoids and limonoids.Results & discussionOverall, along with the commercial orange cultivars ‘Valencia’ and ‘Hamlin’, the HLB-tolerant Poncirus hybrid ‘US SunDragon,’ and the mandarin hybrids Sugar Belle®, FF-5-51-2, and ‘US Superna’ had positive citrus flavor quality. Esters, some sesquiterpenes, along with flavonoids, eriocitrin and quercetin-3-(3R-glucosylrutinoside), were positively correlated with orange flavor while β-ionone and eucalyptol were highly abundant in the mandarins. The flavonoid linarin, was more abundant in Poncirus hybrids with off-flavors than in the Poncirus hybrid ‘US SunDragon’, having high orange flavor. Two mandarin hybrids, FF-5-6-36 and FTP-6-32-67, were not bitter at harvest, but the juice exhibited delayed bitterness after storage at -20°C, which was associated with significant increases of limonin, nomilin, naringenin, and prunin. Interestingly, during freezer storage, a newly identified flavonoid in citrus, tricin-C-hexoside, increased dramatically across all of the genotypes. The identification of disease-tolerant hybrids with satisfactory flavor quality at juicing as well as after storage where delayed bitterness may develop, has great significance for future breeding efforts for fresh fruit or for use in stand-alone juice/juice blends.

Characterization of Citrus × Poncirus Embryo Rescued Hybrids as Rootstock Candidate using Morphological Markers

In generally, there are both of nucellar and zygotic embryos in the citrus seeds after cross breeding. Since genetic variations are very important for success of plant breeding, morphological traits of individuals are evaluated to identify diversity. The present study aimed to characterization of citrus hybrids for new rootstock genotypes based on its morphological characters at seedling stage. A total of 335 putative interspecies hybrids, derived from 3 crosses [Common sour orange (Citrus aurantium L.) × Troyer citrange (Citrus sinensis L. × Poncirus trifoliata L.), Common mandarin (Citrus deliciosa Ten.) × Troyer citrange (Citrus sinensis L. × Poncirus trifoliata L.) and King mandarin (Citrus nobilis L.) × Carrizo citrange (Citrus sinensis L. × Poncirus trifoliata L.)], were observed by their plant morphology. The eight qualitative and five quantitative characteristics of hybrid plants such as seedling growth, leaf and thorniness characteristics were evaluated. The average plant height of the population was found between 70.2 cm and 133.2 cm. The average stem diameter varied between 5.9 mm and 8.0 mm. Hybrid seedlings were separated on the basis of dominant trifoliate leaf marker. There was wide diversity among the accessions with respect to quantitative leaf characters. In terms of leaf division, 268 genotypes have bifoliate and 67 were trifoliate in all combinations, and many intermediate forms were also observed. In addition 66 of the genotypes were thornless while 269 of the genotypes were thorny. The genotype No. 4, has been assessed as triploid, from Common mandarin × Troyer citrange combination, has the longest and dense spines. Morphological markers data were analysed by clustering method to compare similarities of hybrids. The dissimilarity index was observed between 0.004 and 17.318 within three hybridization combinations. The hybrids obtained at 110 days after pollination were more distant relative to each other all hybridization combination.

Metabolomic and quality profiling of Citrus hybrid cv. Yellowball during long-term storage at different temperatures

Metabolomic and quality profiling of Citrus hybrid cv. Yellowball during long-term storage at different temperatures

Fallglo Tangerine

This document is about the citrus hybrid Fallglo. It is the result of a 1962 cross between Bower and Temple made by P. C. Reese. The plant was later selected for further evaluation by Reese and C. J. Hearn (both from the United States Department of Agriculture Horticultural Station in Orlando). Hearn released the variety in 1987. Firs published in September 1993.

Preliminary screening of citrus hybrids for identifying tolerance to Phytophthora nicotianae

Phytophthora is one of the most devastating pathogens affecting citrus production. Breeding resistant genotypes is a durable strategy to manage this pathogen. In this study, in vitro screening of 31 citrus hybrids was done against Phytophthora nicotianae using detached leaf assay. The hybrids tested were developed in twoway crosses between Rangpur lime (RL, Phytophthora sensitive) and sour orange (SO, Phytophthora tolerant). The relative Phytophthora sensitivity was determined from the necrotic lesion area after three days of pathogen inoculation. The parents and hybrids could be differentiated into five resistance classes on the basis of necrotic lesion size and area. Among the 18 SO × RL (SR) hybrids, three hybrids viz. SR 3, SR 10 and SR 19 exhibited the least leaf necrotic length (2.8-3.7 mm), breadth (3.2-3.9 mm) and area (9.2-15.0 mm2), indicating a highly resistant reaction. From the 13 RL × SO (RS) hybrids, RS 6 and RS 22 showed highly resistant reaction by virtue of the least necrotic area (12.7-17.9 mm2). Twelve SR and four RS hybrids produced necrotic lesions of >216.8 mm2 area and were rated as highly susceptible in this assay. The parents- Rangpur lime and sour orange recorded highly susceptible and resistant reaction, respectively, which authenticated the results of the leaf assay. The five highly resistant hybrids identified in this study after ex vitro validation of Phytophthora tolerance should prove helpful in commercial citriculture.

Generation of transgene-free canker-resistant Citrus sinensis cv. Hamlin in the T0 generation through Cas12a/CBE co-editing.

Citrus canker disease affects citrus production. This disease is caused by Xanthomonas citri subsp. citri (Xcc). Previous studies confirmed that during Xcc infection, PthA4, a transcriptional activator like effector (TALE), is translocated from the pathogen to host plant cells. PthA4 binds to the effector binding elements (EBEs) in the promoter region of canker susceptibility gene LOB1 (EBEPthA4-LOBP) to activate its expression and subsequently cause canker symptoms. Previously, the Cas12a/CBE co-editing method was employed to disrupt EBEPthA4-LOBP of pummelo, which is highly homozygous. However, most commercial citrus cultivars are heterozygous hybrids and more difficult to generate homozygous/biallelic mutants. Here, we employed Cas12a/CBE co-editing method to edit EBEPthA4-LOBP of Hamlin (Citrus sinensis), a commercial heterozygous hybrid citrus cultivar grown worldwide. Binary vector GFP-p1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1 was constructed and shown to be functional via Xcc-facilitated agroinfiltration in Hamlin leaves. This construct allows the selection of transgene-free regenerants via GFP, edits ALS to generate chlorsulfuron-resistant regenerants as a selection marker for genome editing resulting from transient expression of the T-DNA via nCas9-mPBE:ALS2:ALS1, and edits gene(s) of interest (i.e., EBEPthA4-LOBP in this study) through ttLbCas12a, thus creating transgene-free citrus. Totally, 77 plantlets were produced. Among them, 8 plantlets were transgenic plants (#HamGFP1 - #HamGFP8), 4 plantlets were transgene-free (#HamNoGFP1 - #HamNoGFP4), and the rest were wild type. Among 4 transgene-free plantlets, three lines (#HamNoGFP1, #HamNoGFP2 and #HamNoGFP3) contained biallelic mutations in EBEpthA4, and one line (#HamNoGFP4) had homozygous mutations in EBEpthA4. We achieved 5.2% transgene-free homozygous/biallelic mutation efficiency for EBEPthA4-LOBP in C. sinensis cv. Hamlin, compared to 1.9% mutation efficiency for pummelo in a previous study. Importantly, the four transgene-free plantlets and 3 transgenic plantlets that survived were resistant against citrus canker. Taken together, Cas12a/CBE co-editing method has been successfully used to generate transgene-free canker-resistant C. sinensis cv. Hamlin in the T0 generation via biallelic/homozygous editing of EBEpthA4 of the canker susceptibility gene LOB1.

The Effects of a New Citrus Rootstock Citrus junos cv. Shuzhen No. 1 on Performances of Ten Hybrid Citrus Cultivars

The importance of rootstock in citrus production lies in its crucial role in determining tree growth, environmental stress tolerance, and fruit quality. Citrus junos Siebold ex Tanaka cv. Shuzhen No. 1, a recently developed rootstock, demonstrates excellent graft compatibility and abiotic stress tolerance. The objective of this study was to assess ten hybrid citrus cultivars grafted onto two C. junos rootstock selections, with the aim of determining the potential for industrial utilization of the new citrus rootstock. All graft junctions are mature and well established. Vigorous growth characterized all ten citrus cultivars on Shuzhen No. 1, with the largest tree’s height reaching 280.33 cm (Wogan scion) and the widest scion’s diameter being 67.52 cm (Chunjian scion). However, the scion-to-rootstock diameter ratio was the lowest at 0.62 (Chunxiang scion). C. junos rootstock selections significantly affected fruit weight (five of ten scions) and fruit color (seven of ten scions) but had negligible impact on peel thickness (nine of ten scions). Furthermore, rootstock type had a significant influence on fruit quality. In conclusion, our findings indicate strong graft compatibility between all scions and C. junos rootstocks, which can impact overall size and fruit quality. Based on these results, Shuzhen No. 1 is recommended as a valuable citrus rootstock.

Tailoring Pectin-PLA Bilayer Film for Optimal Properties as a Food Pouch Material.

This study focuses on developing a biodegradable film using a novel hybrid citrus peel pectin. A bilayer approach with PLA was proposed and optimized using Response Surface Methodology (RSM) to complement pectin films' mechanical and barrier property limitations. The optimized film composition (2.90 g PLA and 1.96 g pectin) showed enhanced mechanical strength with a tensile strength (TS) of 7.04 MPa and an elongation at break (EAB) of 462.63%. In addition, it demonstrated lower water vapor (1.45 × 10-10 g/msPa), oxygen (2.79 × 10-7 g/ms) permeability, and solubility (23.53%). Compared to single-layer pectin films, the optimized bilayer film had a 25% increased thickness, significantly improved water barrier (3806 times lower) and oxygen barrier (3.68 times lower) properties, and 22.38 times higher stretchability, attributed to hydrogen bond formation, as confirmed by FTIR analysis. The bilayer film, effectively protected against UV and visible light, could be a barrier against light-induced lipid oxidation. Moreover, it demonstrated superior seal efficiency, ensuring secure sealing in practical applications. The bilayer pouch containing mustard dressing exhibited stable sealing with no leakage after immersion in hot water and ethanol, making it suitable for secure food pouch packaging.

Upregulation Effect of Citrus Species on Brain-Derived Neurotrophic Factor.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin that plays fundamental roles in neuronal survival and synaptic plasticity. Its upregulation in the brain can effectively prevent and treat central nervous system (CNS) diseases, including depression, Alzheimer's disease (AD), and Parkinson's disease (PD). BDNF is synthesized in various peripheral tissues as well as in the brain and can be transported from peripheral circulation into the brain through the blood-brain barrier. Therefore, foods that upregulate BDNF in peripheral tissues may be beneficial in preventing and treating these CNS diseases. Previously, we revealed that treatment with Chinpi (Citrus unshiu peel) and Citrus natsudaidai increased BDNF levels in the human renal adenocarcinoma cell line ACHN. Here, we evaluated the effects of 21 citrus cultivars on BDNF production in ACHN cells by measuring BDNF levels in the cell culture medium. We found that treatment with peels and pulps of 13 citrus varieties increased BDNF levels in ACHN cells. Treatment with Aurantium, Acrumen, and their hybrids citrus varieties showed a potent BDNF-upregulating effect but not with varieties belonging to Limonellus, Citrophorum, and Cephalocitrus. In addition, treatment with some of those Acrumen and its hybrid citrus species resulted in elevated levels of BDNF transcripts in ACHN cells. These results suggest that peels of many citrus cultivars contain ingredients with a potential BDNF-upregulating ability, which may be novel drug seeds for treating depression, AD, and PD. Furthermore, many citrus cultivars could be used as BDNF-upregulating foods.

Development of fermented beverage with citrus fruit extract using probiotics: impact on antioxidant activity and in vitro digestibility

Redhyang (Citrus hybrid ‘Kanpei’,CHK) is a subtropical citrus species introduced in Korea due to climate change. To enhance the nutritional value and usability of CHK as a processed food product, CHK extract was fermented with four types of commercial starters (YoFlex Harmony 1.0 (YFH), ABY-3 (ABY), YC-X11 (YXC), and YC-180 (YC)), and their antioxidant activities and changes in chemical properties during fermentation were investigated. The consumer acceptance of probiotic beverages containing fermented CHK extracts and their viability and antioxidant activity through in vitro digestion were also elucidated. The enumeration of lactic acid bacteria (LAB) in all samples after fermentation was above 7.60 log colony-forming units (CFU)/mL, with YC exhibiting the highest number after 24 h. Fermented CHK extracts containing higher levels of organic acids, total polyphenols, and flavonoids tended to exhibit higher antioxidant activities. YFH, ABY, and YC showed maximum antioxidant activity at 24 h, whereas YXC showed differences in the types of LAB at 12 h. After in vitro digestion, YXC showed higher antioxidant activity and LAB viability than the control. This result indicates that CHK extract fermented with YXC can increase antioxidant activity, bioactive ingredients, and sensory preference and positively impact the production of probiotic beverages.

Effects of Coconut Water and Banana Homogenate on Shoot Regeneration of Meyer Lemon (Citrus × meyeri)

Meyer lemon (Citrus × meyeri) is a hybrid citrus fruit from the Rutaceae family, originating from China. It is well-known for its distinctive appearance and flavor, as well as its health-nourishing nutrients. Micropropagation is an efficient alternative in the multiplication of plant stocks suitable for the commercial scale. The inclusion of organic additives in culture media has been found to provide a cost-effective option as a plant growth stimulant for in vitro plant development. The current study intends to assess the effects of coconut water and banana homogenate in the regeneration of Meyer lemon. In vitro, shoots were treated in half-strength Murashige and Skoog media fortified with 2 mg/L 6-benzylaminopurine with varying concentrations of coconut water and banana homogenate without sucrose. Results revealed that the treatment of 30% coconut water and 40 g/L banana homogenate resulted in the greatest proliferation of new shoots (3.00 ± 0.873 and 1.57 ± 0.297, respectively), whereas treatment of 40% coconut water resulted in the greatest shoot elongation of 0.239 ± 0.026 cm. The current study suggested the incorporation of coconut water and banana homogenate as potential substitutes for carbon sources and growth stimulants in the regeneration of Meyer lemon.

The Flavor Characterization of ‘Huyou’ (Citrus changshanensis) Essential Oils Extracted by Conventional and Novel Methods

The aroma of citrus is among the most appealing natural flavors. ‘Huyou’ (HY) is a hybrid citrus with a unique flavor compared to grapefruit (GF), but few studies have analyzed its characteristic flavor comprehensively. In this study, we extract six essential oils (EOs) from HY and GF peels by cold pressing (CP) and microwave-assisted hydrodistillation (MADH) and spinning cone column (SCC). Further, the flavor of six EOs was investigated by using flavoromics analysis, including e-nose, GC-MS and GC-O combined with chemometric approaches. The results showed that CP EOs exhibited a stronger citrus characteristic flavor, while MADH and SCC EOs contained more diverse volatiles. A total of 23 key odorants were identified in the GC-O-MS analysis, 12 of which were specific to HY. The flavor wheel and partial least squares regression (PLSR) revealed that floral, sweet and fruity odors were positively correlated with linalool, α-terpineol and geraniol, while fatty, green and woody odors with germacrene D, germacrene B and nootkatone. Additionally, based on orthogonal partial least squares discriminant analysis (OPLS-DA), six aroma-active compounds were screened as aroma markers to distinguish HY from GF; i.e., γ-terpinene, D-limonene, germacrene D, nootkatone, germacrene B and terpinolene. The extraction methods and citrus varieties both impact the flavor characterization of citrus EOs, and our study provides guidance on the extraction and application for citrus EOs.

Fruit Quality Assessment of Novel Hybrid Pummelo × Sweet Orange and Its Molecular Characterization Using Acidity Specific Markers.

There is considerable diversity in newly developed pummelo × sweet orange citrus hybrids. Most hybrids showed lower peel thickness and high juice yield but there is a lack of information on fruit quality parameters and molecular characterization. Therefore, the aim of the current study is to determine the content of antioxidants and properties of the fresh juice of 24 new pummelo × sweet orange citrus hybrids (Citrus maxima [Burm. f.] Osbeck × Citrus sinensis [L.] Osbeck) and the parental genotypes along with molecular characteristics determined using acidity specific markers. The correlation and estimate of inheritance of the fruit juice properties: ascorbic acid, total phenol, total flavonoid, total antioxidant, total soluble solid and sugar contents, pH, titratable acidity, along with sensory evaluation was performed. Molecular characterization of these hybrids was carried out using de novo generated acidity specific simple sequence repeat (SSR) markers. The main constituents of the fruit juice of pummelo × sweet orange hybrids were observed in the range of w(ascorbic acid)=40.00-58.13 mg/100 g, total phenols expressed as gallic acid equivalents w(GAE)=40.67-107.33 mg/100 g, total antioxidants expressed as Trolox equivalents b(Trolox)=2.03-5.49 µmol/g, total flavonoids expressed as quercetin equivalents w(QE)=23.67-59.33 mg/100 g, along with other properties: total soluble solids=7.33-11.33 %, w(total sugar)=2.10-5.76 %, w(reducing sugar)=1.69-2.78 %, w(non-reducing sugar)=0.39-3.17 % and titratable acidity 1.00-2.11 %. The above parameters differed significantly in the fruit juice of the evaluated pummelo × sweet orange hybrids. Considering these parameters, the hybrids SCSH 17-9, SCSH 13-13, SCSH 11-15 and SCSH 3-15 had superior antioxidant properties in terms of these parameters. A higher heritability (≥80 %) was also observed for all juice properties. Molecular characterization of pummelo × sweet orange hybrids showed that >50 % of the hybrids were grouped with medium acidity parents. Both molecular and biochemical parameter-based clustering showed that interspecific hybrids exhibit transgressive segregation with increased antioxidants that help alleviate the health problems. These newly developed pummelo × sweet orange citrus hybrids are a valuable source of high-quality antioxidants for a healthy diet. The identification of trait markers that enable selection at the seedling stage is of great benefit to citrus breeders, as the characteristic features of a mature tree are not yet visible at the juvenile stage.

Harvesting Maturity Assessment of Newly Developed Citrus Hybrids (Citrus maxima Merr. × Citrus sinensis (L.) Osbeck) for Optimum Juice Quality

The assessment of the optimum harvesting stage is a prerequisite to evaluating the performance of new citrus genotypes. The intrinsic and extrinsic fruit quality traits of citrus fruits change throughout their developmental process; therefore, to ensure the highest quality, the fruit must be harvested at an appropriate stage of maturity. The biochemical changes in terms of total soluble solids (TSS), titratable acidity (TA), TSS/TA ratio, BrimA (Brix minus acidity), and ascorbic acid, in addition to the organoleptic acceptability of 16 new interspecific citrus hybrids, were evaluated in New Delhi (India) during the H1-H8 harvesting stage at 15-day intervals to standardize the optimum harvesting stage. The TA and ascorbic acid content were at a maximum level during the early harvesting stage and declined with time, reaching the minimum level in the last harvesting stage. The TSS, TSS/TA ratio, and BrimA values were found to have an increasing trend up to the last stage in most of the hybrids. The juice content shows an inclining trend during the initial harvesting observations, followed by stable juice content and then a decline. The BrimA was found to be a better predictor for consumer acceptability compared to the traditional maturity index TSS/TA ratio and, thus, harvesting maturity. Specific TSS, TA, and BrimA values, in addition to the juice percentage and ascorbic acid content, corresponding to the highest hedonic score, were judged as the optimum harvesting stage indicators for an individual hybrid genotype. Among the interspecific hybrids, SCSH-9-10/12, SCSH-11-15/12, and SCSH-17-19/13 were found to be superior, having better juice acceptability organoleptic scores (≥6.0) and higher juice content (≥40%). Principal component analysis based on fruit physico-chemical traits could be able to distinguish the optimum maturity stage in all of the citrus genotypes.

The effects of different extraction methods on essential oils from orange and tangor: From the peel to the essential oil.

Citrus fruits are largely consumed due to their unique and pleasant aromas. Citrus hybrids have been developed to enhance their flavors and bioactivities. Citrus aroma depends on the composition of the volatile compounds in citrus essential oils (CEOs), which are mostly located in the peels. During the extraction of CEOs, a specific series of chemical reactions occurred depending on the extraction methods (CP, cold pressing; HD, hydrodistillation), leading to variations in the composition of volatile compounds. In this study, the orange and the tangor which is a hybrid between C. reticulata × C. sinensis were investigated to compare the changes in volatile compounds based on the extraction methods. Results showed that the CP-specific volatile compounds were sesquiterpenes, oxygenated monoterpenes, and fatty acid derivatives, while the HD-specific volatile compounds were terpinyl cation derivatives, limonene, and 4-vinylguaiacol. On the other hand, the contents of some volatile compounds ((E)-ocimene, α-terpinene, and α-terpinolene) were affected by the Citrus species rather than by the extraction methods. In particular, during HD, terpinene-4-ol and 4-vinylguiacol, known as off-flavor compounds in citrus juice, were formed more abundantly in the orange than in the tangor. In conclusion, these results provide comprehensive data on essential oils, especially those derived from oranges and tangors, for selecting the appropriate extraction method for obtaining a higher yield and quality of citrus flavor.

Novel edible films fabricated with HG-type pectin extracted from different types of hybrid citrus peels: Effects of pectin composition on film properties

This study investigated the valorization of novel HG-type hybrid citrus pectins derived from three cultivars: Setoka (ST), Kanpei (KP), and Shiranui (SH), and their application as packaging materials. The physicochemical properties of these pectins and their corresponding films were evaluated and compared to commercial citrus pectin. Significant variations were observed in pectin yield (18.15–24.12 %) and other physicochemical characteristics, such as degree of esterification (DE), degree of methoxylation (DM), and monosaccharide composition, among the different cultivars. All hybrid citrus pectins were classified as high-methoxy pectin types (66.67–72.89 %) with typical structural configurations like commercial citrus pectin. However, hybrid citrus pectin films exhibited superior physical properties, including higher mechanical strength, flexibility, and lower water solubility than commercial citrus pectin film, while maintaining similar transparency and moisture content. Additionally, the films displayed smooth and uniform surface morphology, confirming their excellent film-forming properties. Correlation analysis revealed that DE positively influenced mechanical properties (r = 1.0). Furthermore, the monosaccharide composition of pectins showed strong relationships (r = 0.8–1.0) with the film's mechanical and barrier properties. These findings highlight the potential of hybrid citrus pectin as potential packaging material, and the knowledge of the structure-function relationship obtained in this study could be useful for the tailored modification of citrus pectin-based packages.

Species of Diaspididae (Hemiptera: Coccomorpha) on citrus in southern Lao PDR, new records for Việt Nam, and revised records for Indochina

The Diaspididae (Hemiptera: Coccomorpha), also known as armoured scale insects, is a diverse family of obligate, phytophagous parasites that include major economic pests of terrestrial plants. There are over 2,700 diaspidid species, about 116 of which have been recorded worldwide feeding on species and hybrids of citrus. Citrus is widely cultivated in the Lao People’s Democratic Republic (Lao PDR) and has been promoted by the country’s government as a poverty alleviation crop. However, little is known about the pest and disease issues faced by farmers of this crop. To help address this, in 2018 we conducted surveys of diaspidids on citrus in southern Lao PDR and found 14 species infesting citrus growing in orchards and home gardens. Identifications were based on morphological features of adult females and 28S, COI and COII DNA sequences. An identification key to the species based on morphological characters is provided. Records of diaspidid species on citrus in Myanmar, Thailand and Cambodia are updated, and records from Peninsular Malaysia provided. The first record on citrus of a Pseudaulacaspis species in Indochina is reported from Việt Nam, and an undescribed species of Pseudaonidia is recorded on citrus in Lao PDR.

First report of grapefruit rot caused by Colletotrichum gloeosporioides and C. karsti in France.

The grapefruit (Citrus paradisi) is a citrus hybrid tree (C. maxima & C. sinensis). Due to nutritional value and its bioactive compounds, the fruits are recognized as a functional food, valued as promoting health. French grapefruit production is low (7.5 Kt/year) but is confined to a restricted area in Corsica and benefits from a quality label, the economic impact of its cultivation being therefore locally significant. Since 2015 previously unreported symptoms have been repeatedly observed on grapefruits in more than half of the orchards in Corsica, with an incidence of 30% of fruits altered. Brown to black circular spots were observed on fruits and leaves, surrounded by chlorotic halos on the latter. On the mature fruit, lesions were round, 4 to 10 mm in diameter, brown and dry (e-Xtra 1). Although the lesions are superficial, the fruits cannot be marketed due to constraints linked to the quality label. 75 fungal isolates were obtained from symptomatic fruits or leaves collected in Corsica (in 2016, 2017, and 2021). Cultures obtained after 7 days on PDA at 25°C, were white to light grey in colour, forming concentric rings or dark spots on the agar surface. We did not observe any notable difference among the isolates except some evolved towards a more marked grey. Colonies tend to form a cottony aerial mycelium and orange conidial masses appear with age. The conidia were hyaline, aseptate, cylindrical with ends rounded, and measured 14.9 ± 0.95 µm length and 5.1 ± 0.45 µm width (n = 50). Cultural and morphological characteristics were similar to those described for C. gloeosporioides s. lat. or C. boninense s. lat. (Weir et al. 2012 ; Damm et al. 2012). Total genomic DNA was extracted from all isolates, and the ITS region of rDNA was amplified with ITS 5 & 4 primers, then sequenced (GenBank Accession Nos. OQ509805-808). For 90% of isolates GenBank BLASTn results were 100% identical to C. gloeosporioides isolates sequences, whereas for other isolates the resulting sequences were 100% identical to C. karsti or C. boninense isolates sequences. Four strains (three C. gloeosporioides with light colour differences, in order to see if there was diversity among isolates of C. gloeosporioides s. lato ; and one C. karsti) were further characterized by sequencing partial actin [ACT], calmodulin [CAL], chitin synthase [CHS-1], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], β-tubulin 2 [TUB2], for all strains ; glutamine synthetase [GS], Apn2-Mat1-2-1 intergenic spacer and partial mating type (Mat1-2) gene [ApMAT] for C. gloeosporioides s. lat., and HIS3 for C. boninense s. lat. (Weir et al. 2012 ; Silva et al, 2012) (GenBank Accession Nos. OQ509805-808 & OQ507698-724). Multilocus phylogenetic analyses carried out with the obtained and Genbank available sequences confirmed that 3 isolates (UBOCC-A-116036, -116038, & -116039) clustered within C. gloeosporioides s. s., while the other (UBOCC-A-116037) clustered within C. karsti (e-Xtra 2) 'Star ruby' grapefruits were surface sterilized then wound-inoculated with 20 μl of a conidial suspension (105 conidia ml-1) of UBOCC-A-116036 & 116037 isolates or 20 μl sterile water for control (ten fruits for each isolate or control). After 10 days incubation at 20°C, symptoms, identical to those initially observed, developed around the inoculation point, while controls inoculated with water remained symptomless. Fungal colonies re-isolated from the lesions were morphologically like the original isolates. Recently, various infections caused by some Colletotrichum sp. have strongly compromised citrus production in different Mediterranean countries: ie Italy (Aiello et al. 2015), Portugal (Ramos et al. 2016), Tunisia (Ben Hadj Daoud et al. 2019), Turkey (Uysal et al. 2022). In these studies, C. gloeosporioides s. s. and C. karsti were identified as the causal agents. These two species were the predominant Colletotrichum sp. associated with Citrus and allied genera in Europe (Guarnaccia et al. 2017). To our knowledge, our study is the first report of C. gloeosporioides and C. karsti causing anthracnose on grapefruit in France, which confirms the incidence of these two pathogens on the Mediterranean rim. Given the economic importance of citrus cultivation in the Mediterranean region, the presence of Colletotrichum spp. should deserves to be monitored, and a control strategy should be considered.

Portable LWNIR and SWNIR spectroscopy with pattern recognition technology for accurate and nondestructive detection of hidden mold infection in citrus

An accurate and nondestructive detection method of hidden mold infection in citrus was established based on portable near infrared diffuse reflectance spectroscopy (NIRDRS) and chemometric methods. Penetrability of NIRDRS light on the peel of Chunjian hybrid citrus was studied. The results show that NIRDRS light can penetrate the peel to a certain extent, while the penetrability of short-wave near infrared (SWNIR) was better than that of long-wave near infrared (LWNIR). The identification models of hidden mold infection were established with five pattern recognition methods combined with different wavelength bands. The results show that the identification models of LWNIR were much better than those of SWNIR. 100% identification accuracies of LWNIR were obtained with soft independent pattern classification (SIMCA), support vector machine (SVM), partial least squares discriminant analysis (PLS-DA) and the optimized pretreatment methods. In addition, the developed models were further validated by the external validation set collected one month later.

Molecular cytogenetic maps provide new insights into the differences between hybrid Citrus microcarpa and lemons

Citrus microcarpa is one of the most popular species for flavoring and industrialized juice production, making it an increasingly important economic crop. At present, C. microcarpa has been mistakenly regarded as a type of lemon in China. The lack of a simple identification method for hybrid C. microcarpa is the major obstacle limiting its comprehensive recognition and utilization. In this study, firstly nine pairs of chromosomes were marked by eight oligo pools (OPs) in C. microcarpa, C. aurantiifolia, and C. limon, which showed that oligonucleotide (oligo) sequences are highly conserved within the genomes of the citrus family, suggesting that eight OPs and nine chromosome painting probes (CPPs) can be used as universal probes for the identification of each chromosome in citrus plants. Then we developed a method for the identification of C. microcarpa by using fluorescence in situ hybridization (FISH) mapping and Chromoycin A3 (CMA) staining. Moreover, we constructed the molecular cytogenetic maps containing the physical positions of eight OPs, 45S rDNA, 5S rDNA, CL, CMA banding and fragile sites in C. microcarpa, C. aurantiifolia, and C. limon, which visualize the significant differences among the three Citrus species. Combining the highly different morphological and cytological characteristics of C. microcarpa with C. aurantiifolia and C. limon, it is clear that C. microcarpa is a Citrus hybrid species, not a lemon species. Overall, this study provides a better understanding of C. microcarpa from the molecular cytogenetic perspective.