Moroccan Citrus Research Articles

Quantitative and qualitative characterization of Moroccan citrus lime accessions

Quantitative and qualitative characterization of Moroccan citrus lime accessions

Moroccan Citrus clementina Peels: Optimization of Pectin Extraction and Determination of Chemical and Functional Properties.

Citrus peels are considered a rich source of valuable biomolecules. Pectin is a polymer of polysaccharide acid and is composed of galacturonic acid monosaccharides. In this study, response surface methodology was used to optimize pectin extraction from Citrus × clementina Hort. ex Tan. (Rutaceae) peels using citric acid as an extraction solvent. The effect of the parameters conditioning the extraction process and pectin yield (pH level, temperature, extraction time, solid/liquid ratio, and raw material particle size) was investigated using a Box-Behnken design. The quality of the extracted pectin was assessed both chemically (moisture, ash, protein, and carbohydrate content) and functionally (gelling power and emulsifying activity). According to the screening experiment, the pH level, temperature, and particle size were the main factors influencing the pectin yield. The adjusted mathematical model enabled us to plot response surfaces in order to determine the optimal extraction conditions. The highest production yield of pectin (26.6%) was obtained at the optimal conditions of pH = 1.5, temperature = 100 °C, and particle size = 0.1 mm for an extraction time of 30 min. Compared to the predicted value of 26.6%, the experimental extraction yield of C. clementina was about 21.4% of pectin. Concerning the functional properties, the extracted pectin had a high gelling power of 164 ° SAG and an emulsifying activity of 38.5%.

Chemical composition, antibacterial and antioxidant properties of three Moroccan citrus peel essential oils

Citrus peels, which make up a significant portion of the entire fruit and are frequently rejected by nature and have a detrimental influence on the environment, are the focus of this study. These barks can be a highly valuable source of plant material. Three different citrus species C. limonum, C. reticulata, and C. Paradisi had their essential oils extracted from their peels(by-products) to create these citrus co-products (essential oil).The findings demonstrate how rich these citrus species are in essential oils. Limonene is the predominant component for the three, accounting for percentages of 56.3%, 76.5%, and 71.7% for C. paradisi, C. reticulata, and C. limonum, respectively, in their chemical composition as determined by GC/MS analysis. Their antioxidant activity was assessed using DPPH and validated using the FRAP assay. Furthermore, essential oils antimicrobial efficacy against five pathogenic strains: Escherichia coli, Enterococcus feacalis, Staphylococcus aureus, Candida albicans, and Sacharomyces cerevisiae, demonstrates significant antibacterial potential, particularly against Candida albicans.

Comparison of Serological and Molecular Methods for Detection of Spiroplasma citri in Moroccan Citrus-Growing Areas.

Spiroplasma citri, a helical motile, wall-less, and cultivable microorganism of the class Mollicutes, is the agent of the citrus stubborn disease. There is currently a lack of data about the presence of this pathogen in Moroccan citrus orchards. This study aims to validate serological and molecular methods for routine S. citri diagnosis in Moroccan citrus groves. To provide an update on the present status of the outbreak of the pathogen in Moroccan citrus orchards, a survey of S. citri was conducted in the main citrus-growing regions of Morocco. A total of 575 leaf samples were collected from citrus trees with symptoms attributable to S. citri infection. Samples were collected during 2020 and 2021 from 23 citrus orchards. The presence of S. citri was tested in all samples using the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Using this method, 57 samples were found to be infected with S. citri, 41 samples had doubtful results, and the remaining samples were negative. To corroborate the results of the DAS-ELISA test, 148 samples were chosen for additional molecular testing using conventional polymerase chain reaction (PCR) and real-time PCR (qPCR) based on specific primer pairs targeting three different genes (putative adhesion-like gene P58, putative adhesion gene P89, and spiralin gene). Using primers that target the putative adhesion-like gene P58, S. citri was detected by conventional and real-time PCR amplification from plant tissue with differing degrees of specificity. The results allowed us to determine the incidence of S. citri in all Moroccan citrus orchards, with a wide range of positive samples varying from 6.5% to 78%, and to show that molecular tests, particularly real-time PCR assays that target the putative adhesion-like gene P58, are the most sensitive for making an accurate diagnosis of S. citri. Indeed, the real-time PCR with P58-targeting primers yielded positive results from all positive and doubtful ELISA samples as well as some negative samples, with an OD value close to 1.5× times healthy samples, thus demonstrating a high sensibility of this technique.

Machine Learning Algorithms Applied to Semi-Quantitative Data of the Volatilome of Citrus and Other Nectar Honeys with the Use of HS-SPME/GC-MS Analysis, Lead to a New Index of Geographical Origin Authentication.

The scope of the current study was to monitor if semi-quantitative data of volatile compounds (volatilome) of citrus honey (ch) produced in different countries could potentially lead to a new index of citrus honey authentication using specific ratios of the identified volatile compounds in combination with machine learning algorithms. In this context, the semi-quantitative data of the volatilome of 38 citrus honey samples from Egypt, Morocco, Greece, and Spain (determined by headspace solid phase microextraction coupled to gas chromatography mass spectrometry (HS-SPME/GC-MS)) was subjected to supervised and unsupervised chemometrics. Results showed that honey samples could be classified according to the geographical origin based on specific volatile compounds. Data were further evaluated with additional nectar honey samples introduced in the multivariate statistical analysis model and the classification results were not affected. Specific volatile compounds contributed to the discrimination of citrus honey in different amounts according to geographical origin. These were lilac aldehyde D, dill ether, 2-methylbutanal, heptane, benzaldehyde, α,4-dimethyl-3-cyclohexene-1-acetaldehyde, and herboxide (isomer II). The numerical data of these volatile compounds was summed up and divided by the total semi-quantitative volatile content (Rch, Karabagias-Nayik index) of citrus honey, according to geographical origin. Egyptian citrus honey had a value of Rch = 0.35, Moroccan citrus honey had a value of Rch = 0.29, Greek citrus honey had a value of Rch = 0.04, and Spanish citrus honey had a value of Rch = 0.27, leading to a new hypothesis and a complementary index for the control of citrus honey authentication.

Evaluation of Amblyseius swirskii Athias-Henriot and Amblyseius andersoni (Chant) (Acari: Phytoseiidae) as biological control agents of Eutetranychus orientalis (Klein) (Acari: Tetranychidae) on citrus

Amblyseius andersoni and Amblyseius swirskii are used to control the citrus pest Eutetranychus orientalis. Field experiments were conducted in Marrakech region for two months (November and December) in 2018 to evaluate the control effect of these predator species releases. In the control of E. orientalis population, Amblyseius andersoni with100 mites/tree reduced the densities under 3 motiles/leaf, while rates of 25 and 50 mites/tree these were maintained between 8.93 and 4.76 motiles per leaves, and between 12.16 and 4.33 eggs per leaves 5 and 6 weeks after treatment (WAT). Nevertheless, Amblyseius swirskii showed moderate performance, especially with 100 mites/tree densities ranged between 3.45 and 3.52 motiles per leaves, and between 14.8 and 9.46 eggs per leaves 5 and 6 WAT. The combination of both predators showed no significant difference to the former results. In summary, the two predators showed the potential to control E. orientalis in Moroccan citrus.

Physicochemical and functional characterization of pectin extracted from Moroccan citrus peels

Three Moroccan citrus peels, such as orange, lemon, and bigarade, were chosen to extract the pectin using Box Behnken Design. At a temperature of 90 °C, pH = 1.5, and extraction time = 120 min, a high pectin yield was recorded with 28.57, 26.86, and 26.55% for lemon (LPP), bigarade (BPP), and orange (OPP) peel pectins respectively. The extracted pectins have a less branched linear structure with high GalA contents for BPP (86.0 mol%), LPP (82.0 mol%), and OPP (79.0 mol%), and a high degree of methyl esterification (>74%), but a low degree of acetylation (<4%). A narrow and homogeneous distribution was recorded with a higher average Mw for LPP (90.0 kDa) than OPP (88.0 kDa) and BPP (80.0 kDa). One gram of Moroccan citrus pectin could hold more than 23.0 g of water. The high emulsifying activity was recorded for the three citrus pectins (∼50%), with higher emulsion stability for BPP pectin at 4 °C (94.42%). Furthermore, all extracted pectins exhibit a significantly non-Newtonian pseudoplastic behavior when the concentration increases. At 3% concentration, the apparent viscosity of BPP was higher than that of OPP and LPP with 40.26, 13.45, and 6.85 Pa s, respectively.

Seasonal trend of Eutetranychus orientalis in Moroccan citrus orchards and its potential control by Neoseiulus californicus and Stethorus punctillum

Citrus brown mite, Eutetranychus orientalis (Klein), known as major pest of citrus crops in different countries, was considered as a quarantine pest in Morocco. This pest is currently among the most potential enemies of Moroccan arboriculturists following the increasing ineffectiveness of the majority of acaricides used in conventional orchards. The present study was designed to collect and morphologically identify specimens from 31 orchards in the five main production regions of Morocco; to study the population dynamics of this pest in Taroudant as well as the molecular identification of a Phytoseiid considered as a potential predator using the mitochondrial 12S rRNA gene. Morphological description confirmed that the collected adult females from all orchards in the five regions belong to E. orientalis. Thus, this exotic pest has invaded all citrus growing regions since its introduction. In the Souss region, two significant peaks of population outbreaks of this pest were observed. A first peak from November until the end of December 2017 and in winter, a second peak is recorded in early autumn 2018. Thereafter, mite infestations remained at lower levels throughout the summer. In addition, it appears that eggs represented between 50 to 88.7% of population in spring and winter seasons respectively, but percentage of adult females does not exceed 2.3% in autumn and winter seasons. Adult females of Neoseiulus californicus, larvae of Stethorus punctillum, were more abundant and very active on orange leaves and weeds during period of observation. Molecular analysis using 12S ribosomal RNA gene confirmed that the best query coverage 99.23% (E-value = 5e-176) with 99% of recovery identical to N. californicus. The phylogenetic tree obtained from comparison of the specimens’ generated DNA sequences with other individuals from Neoseiulus genus revealed the relationship with species N. californicus. These results suggest that orchard management will favour the plant cover associated with citrus trees and thus ensure sustainability, diversity and high density of phytoseiides and other predatory insects. This auxiliary fauna, like N. californicus, S. punctillum, will be able to control spider mites, as well as other phytophagous insects and keep them below damaging levels in citrus orchards in the Souss region.

Evaluation of in vivo wound healing activity of Moroccan Citrus reticulata peel extract

BackgroundCitrus reticulata is one of the most cultivated fruit with great benefits for humans in the world. Citrus reticulata peel has several biological activities within them hypoglycemic, hepatoprotective, antimicrobial and antioxidant. The present study emphasizes on the in vivo wound healing and in vitro antimicrobial and antioxidant activities of Citrus reticulata peel extract.MethodsForty albino mice (23–29 g) of either sex were divided into four groups. The test groups were treated with experimental ointment (0.5% and 10% of methanolic extract), negative control were treated with Vaseline and the positive control were treated with silver sulfadiazine. Burn wounds were induced on dorsal area of mice bodies. Wound area measurement was carried out every day during 22 days. Biochemical screening was performed to identify possible compounds. Antioxidant and antimicrobial activities were also determined.ResultsSignificant wound healing activity was observed with topical application of Citrus reticulata peel extract. Wound area reduction at day 16 of treatment was 100% for both treated groups (0.5% and 10%) when compared to positive and negative control it was 100% and 98.32% respectively at day 22. Furthermore higher rate of wound contraction (100% on 16th day) was observed for both treated groups.The result of biochemical screening showed that C. reticulata peel is characterized by highest amount of total polyphenols (13.19 mg/g), flavonoids (4.07 mg/g), vitamin C (13.20 mg/g), carotenoids (0.032 mg/g) and lowest content of macronutrients (Proteins: 0.40%, reducing sugars: 7.21%; lipids: 1.5%). Additionally C. reticulata peel exhibited remarkable antioxidant activity using DPPH and phosphomolybdate methods as well as the extract possess antimicrobial effect against pathogen bactria.ConclusionThe findings from this research indicated that Citrus reticulata peel extract is effective in inhibiting the growth of pathogen bacteria and could be of therapeutic potentials for wound healing.

Beneficial insect community of Moroccan citrus groves: assessment of their potential to enhance biocontrol services

In citrus groves, beneficial insects that reduce abundance of pests are considered a key component of integrated pest management strategies. The aim of this article was to assess the biodiversity of parasitoids and predators in citrus orchards in Moroccoto facilitate future investigations on their potential as biocontrol agents. Data of 105 citrus beneficial insects were gathered and summarized in a data matrix. Variables such nature, target pests, type, establishment, and efficacy were assessed. More than two-thirds of parasitoids and predators species identified in citrus groves of Morocco (105 species) are native (> 70%). Both groups represent only a small fraction of the introduced species. The mostly attack armored scale insects (Diaspididae) and aphids (Aphididae). The ladybeetle Rodolia cardinalis (Mulsant) (Coleoptera: Coccinellidae) is the first beneficial species introduced in 1921 to the Moroccan citrus orchards to control the cottony cushion scale Icerya purchasi (Maskell) (Hemiptera: Monophlebidae). Major introductions of these parasitoids and predators were carried out during the ninetieth to control the main citrus pests whereas they were accidentally introduced. These purposely introduced species are mainly Aphelinidae, Encyrtidae, Eulophidae, Coccinellidae, and Phytoseiidae. Whereas a high proportion of the introduced beneficial insects was established and no species have been reported to be harmful to this date. Considering only the introduced species used in classical biological control context, about 20 and 40% of them are considered as effective or partially effective, respectively.

Evaluation of the Impact of Partnership Program on the Technical and Economic Efficiency of Irrigation Water Use for Moroccan Citrus Farms

A public-private partnership (PPP) in irrigated area is a mode of collaboration and governance that allows the Moroccan Government to entrust the design, financing, construction, operation and maintenance of irrigation equipment to a private enterprise. In this research, we want to estimate technical efficiency of farms, the technical and economic efficiency of irrigation water use, analyzing the determinants of each of these aspects. The sampling was undertaken using the propensity score matching. The estimation of the farms’ technical efficiency was carried out using FRONTIER 4.1 software and a stochastic parametric method with the functional form Translog. The transition from an output orientation to an input orientation in order to estimate the technical and economic efficiency scores of the irrigation water was carried out through the use of the Reinhard derivative. The determinants of these aspects were analyzed with the Gnu Regression, Econometrics and Time series Library (Gretl) software using the Tobit model. A survey was carried out among 130 citrus growers (65 pairs). The average of the technical efficiency increased from 62% in 2009 to 72% in 2016. This improvement is explained by the presence of highly qualified employed who assure the management and the technical supervision of the workers on the farms. Conversely, the analysis shows that the PPP irrigation program has had no impact on the farms’ technical efficiency scores. On the basis of the results, it is appropriate to focus on several aspects in order to improve the different types of efficiencies. Indeed, the Moroccan Government must ensure the aspect of the technical supervision of farmers in the two perimeters, which will better help to control the technical conduct of citrus farms.

Reactive and Robust Planning of Moroccan Citrus Chain Based on Multi-agent System and Performance Indicators

As part of improving services done for various clients in all Moroccan areas, Moroccan exportation group of fruits and vegetables in collaboration with their packaging units and producers, tends to cooperate in order to face with international competitiveness. Indeed, the complexity of networks of partners has led policy-makers to implement new techniques and tools to help control different processes. For this reason, the implementation of a permanent monitoring of different operations ranging from production, packaging, and distribution of perishable products has become paramount. This article aims to propose a model of multi-agent citrus supply chain, based on indicators for monitoring and evaluation of performance of its logistics systems, in order to build a new independent, robust and responsive chain, and to optimize and control the flow of materials and information between the different actors and stakeholders of the chain. review summarizing existing models of modeling supply chains; second, a presentation of the principle of distributed modeling based on multi-agent approach and its impact on our case studied system; third, we project the model selected on Moroccan citrus supply chain; finally, we proposed a model of decision that is based on mixed architecture for reactive and robust chain compared to disturbances and uncertainties. 2. State of the Art

CITRUS ROOTSTOCKS IN MOROCCO: PRESENT SITUATION AND FUTURE PROSPECTS

Until the last decade, the Moroccan citrus industry has relied exclusively on sour orange as a rootstock. However, rootstock trials were initiated in the 1960s at the experimental farms of INRA. The major selection traits targeted at that time included yield and fruit quality, compatibility with clementine varieties and tolerance to Phytophthora gummosis, salt and limestone soils. In spite of the outstanding results of some rootstocks revealed by these trials, sour orange continues to be the main rootstock of choice of citrus farmers. Since the year 2000, after a long and intense debate among citrus industry stockholders, a vivid action was initiated to diversify citrus rootstocks in new plantings. While sour orange remained in use, trifoliate hybrids (Carrizo, Troyer, and C-35 citranges), C. volkameriana, and C. macrophylla started to increase in percentage, especially in large scale nurseries. In 2007, during a sanitary survey in the Loukous region Northwest of Morocco, Citrus tristeza virus (CTV) was detected at high incidence in many orchards. An intense effort is actually being devoted to promote the use of CTV tolerant rootstocks. Figures of how each one of these rootstocks has developed during the last 8 years are presented. A preliminary assessment of the impact of the rootstock shift on yield, quality and market reaction is discussed.

The Ability of the Antagonist Yeast Pichia Guilliermondii Strain Z1 to Suppress Green Mould Infection in Citrus Fruit.

In previous studies it was shown that Pichia guilliermondii strain Z1, isolated from healthy Moroccan citrus Valencia-Late oranges, was effective against Penicillium italicum. Here the effectiveness of strain Z1 was assessed against Penicillium digitatum, the causal agent of green mould, under different temperature (5-25°C) and relative humidity (RH) (45-100%) regimes for its reliable and large-scale application in packinghouse. All main effects and interactions were significant (P<0.0001). In the pathogen control, the largest lesion diameter was at an RH range between 98 and 100%, regardless of the incubation temperature. The efficacy of strain Z1 was not dependent on the environment and reduced disease incidence by >80%. Its applications as a formulated product significantly reduced the incidence of infected fruit (55%) and the percentage of infected wounds (47%) compared to the only pathogen control treatment. However, disease control with formulated product was significantly less than that obtained with thiabendazole (30%) or strain Z1 culturable cells (35%). These results highlight that strain Z1 is an effective biological control agent for control of green mould under varying environmental conditions, and control may be optimized by combining its use with other environmentally-safe post-harvest treatments or improved formulation.

Analyse et modélisation de la chaîne agrumicole marocaine basée sur le système multi-agents et indicateurs de performance : planification réactive et robuste

As part of the improvement of the services addressed to the various customers in all the Moroccan production zones, the Moroccan fruit and vegetable export groups in collaboration with their packaging units and their producers tend to cooperate with each other in order to face international competitiveness. Indeed, the growing complexity of partner networks has prompted decision-makers to put in place techniques and tools contributing to the control of the various processes. For this, the establishment of a permanent monitoring of the various operations ranging from production, packaging, to the distribution of perishable products has become essential. This article aims to propose a multi-agent modeling of the citrus supply chain based on indicators for monitoring and evaluating the performance of its logistics systems. This with the aim of building a new autonomous, robust and responsive chain, thus making it possible to optimize and control the flow of materials and information between the various actors and stakeholders in the chain. The article is presented along four major axes: first, a literature review summarizing the different existing models for the modeling of supply chains. Then, a presentation of the principle of distributed modeling based on the multi-agent system and its application on our case study. On the third axis, we have projected the principle of the model chosen on the Moroccan citrus chain. Finally, we proposed a decision-making model based on a mixed architecture allowing a reactive and robust chain with regard to disturbances and uncertainties.

Impact of some alternative methods to chemical control in controlling aphids (Hemiptera: Sternorrhyncha) and their side effects on natural enemies on young Moroccan citrus groves

Aphid control in Moroccan citrus orchards is based mainly on carbamate and neonicotinoid sprays, especially methomyl and imidacloprid. The extensive use of these insecticides may have side effects on natural enemies and environment quality and raises human health concerns. This research aimed to assess the control of aphids with insecticidal soap, kaolin and augmentative biological control using the indigenous predator Adalia decempunctata L. (Coleoptera: Coccinellidae). The insecticides were applied and the predators were released in April 2009 and 2010. Under field experimentation, the methomyl and imidacloprid foliar pulverization were very effective against aphids. In contrast, the insecticidal soap and kaolin application were less efficient while A. decempunctata adults were effective only in the first week after release. The side effects on beneficial insects were also assessed and discussed. The possibility of employing A. decempunctata in an integrated pest management package in citrus groves is discussed in relation to effectiveness and side effects on beneficial arthropods.

Influence of growth regulators on callus induction from embryos of five citrus rootstocks

Objective: The aim of this work is to develop a protocol for callus induction of Moroccan Citrus rootstocks. Methodology and results: Callus cultures were initiated from embryos explants of five Citrus rootstocks (Cleopatra mandarin , Rangpur lime , Citrus volkameriana , Trifoliate orange , Citrus aurantium ) on Murashige and Tucker (MT) and Tucker (MT) basal media. Different concentrations of growth regulators were tested in order to obtain the best callus formation: 2, 4-dichloro-phenoxyacetic acid (2, 4-D) in combination with benzylaminopurine (BAP) at five levels (0, 0.5, 1, 2 and 3mgL-1) were used in this study. It was found that growth regulator concentration had a significant effect on the callus induction, the callus growth and callus physical appearance. The highest frequency of the callus induction rate (100% and 83%) was observed with two combinations of 2, 4-D/BAP: 1 / 0, 5 and 2/1 (mgl-1). Medium containing only BAP (1mgl-1) resulted in the formation of large numbers of roots. Also, the callus induced on MT medium containing only 2, 4-D (1mgl-1) was brown in color and of low quality compared to that produced on MT media containing 2, 4-D/BAP. There was no callus formation on MT basal medium without growth regulators. Also, the callogenesis depended on the genotype. It was maximal for Cleopatra mandarin followed by Rangpur lime and Citrus volkameriana , and then Citrus aurantium and trifoliate orange . Conclusion and application : Passing through callus is paramount in vitro selection that plant tissue is an important source of genetic variability. The explants and exogenous phytohormones proved of paramount importance for the success of the callus and the formation of somatic embryos and their development.

Shelf-life and quality evaluation of clementine following a combined treatment with γ-irradiation

In order to enhance the shelf-life of a late variety of Moroccan Citrus clementina (Nour), ionizing treatments were applied at 0.3 kGy, as well as washing (cold water) and waxing treatments. It has been found that, despite the irradiation treatment, the washing and waxing treatment do not improve the quality of C. clementina, but rather result in peel injury. Finally, sensory evaluation confirmed that irradiation had no detrimental effect on the quality of clementines.

Effect of gamma-irradiation combined with washing and waxing treatment on physicochemical properties, vitamin C, and organoleptic quality of Citrus clementina Hort. Ex. Tanaka.

To enhance the shelf life of a late variety of Moroccan Citrus clementina (Nour), ionizing treatments were applied at 0.3 kGy, as well as washing (cold water) and waxing treatments. It has been found that, despite the irradiation treatment, the washing and waxing treatment do not improve the quality of C. clementina, but rather result in yellower peels, peel injury, and reductions of vitamin C content, acidity, and soluble solids. However, gamma-irradiation alone enhanced significantly (p <or= 0.05) the level of vitamin C and the total phenolic content and maintained the color of the C. clementina during the entire storage period (49 days at 3 +/- 1 degrees C and 84% relative humidity). Finally, sensory evaluation further confirmed the beneficial effect of gamma-irradiation. Irradiated clementines were found to be sweeter. Also, the sensorial score of irradiated (I) and washed, waxed, and un-irradiated (LC) fruits was maintained over 7 days during 21 days as compared to 14 days for unwashed, unwaxed, and un-irradiated (C) and for washed, waxed, and irradiated (LCI) fruits.

Citrus variegation virus : Molecular Variability of a Portion of the RNA 3 Containing the Coat Protein Gene and Design of Primers for RT-PCR Detection

The coat protein gene and part of the intergenic region of the RNA 3 of several isolates of Citrus variegation virus (CVV) producing either infectious variegation or crinkly leaf symptoms were amplified by RT-PCR, cloned and sequenced. Some isolates were composed of a mixture of sequence variants. The coat protein gene appeared to be highly conserved (lowest similarity among all CVV sequences 93%), especially at the N-terminal, indicating at the molecular level that both types of symptoms are indeed produced by the same virus species. No relationship could be found with the geographic origin. Sequences obtained from isolates producing infectious variegation clustered in a different branch of a dendrogram than those originating from crinkly leaf symptoms. Both clusters could further be distinguished by two parsimonious sites in the coat protein gene. In the short stretch of the intergenic RNA 3 region analysed, a stable hairpin exists in addition to the previously reported hairpin that constitutes the core promoter for the RNA 4 transcription. This second hairpin could also be recognised in the other subgroup 2 Ilarviruses. Surprisingly, at the nucleotide and amino acid levels and in the secondary features of the intergenic region, CVV appeared closer to the other subgroup 2 Ilarviruses than to Citrus leaf rugose virus to which it is serologically related and has been considered to be evolutionary related. Using primers designed for the conserved regions, the virus was detected with a prevalence of 25% and 13% in Portuguese and Moroccan citrus collections. A group of RT-PCR positives was further confirmed by ELISA and biological indexing.