Antioxidative, phytochemical and antimicrobial analysis of juices of eight citrus cultivars grown in Pakistan

Comparative nutritional analysis of citrus varieties cultivated in Pakistan has not been reported. Citrus is consumed all over the world due to its taste and also has pharmacological components. The present investigation evaluated the antioxidant, reducing power, total flavonoids and phenolics, DPPH free radical scavenging, protein kinase inhibition, and the antimicrobial activities of eight Pakistani citrus varieties. Grapefruit showed maximum total antioxidant potential (77 µg AAE/100 mg), followed by Kinnow and Shakri. Khatai showed maximum reducing power potential (69.6 µg AAE/100 mg) while Shakri and Grapefruit trailed it. All the varieties showed significant DPPH free radical scavenging activity. Maximum total phenolics in citrus juice were found in Shakri and Kinnow; 26.2 and 25.9 µg GAE/100mg, respectively. Variation in total flavonoids content was observed as Kinnow>Grapefruit>Shakri>Khatai. All the citrus juices showed mild to moderate antibacterial activity, while Mosambi and Malta contained potent antifungal components. HPLC analysis of citrus juices revealed that catechin was present in all citrus genotypes except Kinnow. The study concludes that citrus juices contain strong antioxidative potential, bear protein kinase inhibitors and can be used as cancer chemoprevention and supportive nutrition.

Comparative Metabolomics Analysis of Citrus Varieties

Many citrus varieties are hybridized to improve their quality and to overcome the effects of climate change. However, there is limited information on the effect of the chemical profiles of hybrid varieties on their quality. In this study, we analyzed 10 citrus varieties and evaluated the correlation with their general characteristics and antioxidant activities. Chemical profiles, including the contents of sugars, organic acid compounds, flavonoids, limonoids, and carotenoids, which are related to taste, color, and health benefits, were significantly different depending on the citrus varieties, leading to different antioxidant capacities and general quality parameters. Based on these data, the correlations were investigated, and 10 citrus varieties were clustered into four groups—Changshou kumquat and Jeramon (cluster I); Setoka (cluster II-1); Natsumi, Satsuma mandarin, and Navel orange (cluster Ⅱ-2); Kanpei, Tamnaneunbong, Saybyeolbong, and Shiranui (cluster II-3). Moreover, a metabolomic pathway was proposed. Although citrus peels were not analyzed and the sensory and functional qualities of the citrus varieties were not investigated in this study, our results are useful to better understand the relationship between citrus quality and metabolite profiles, which can provide basic information for the development and improvement of new citrus varieties.

Rapid evaluation of the resistance of citrus germplasms against Xanthomonas citri subsp. citri

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus bacterial canker (CBC), one of the most devastating citrus diseases. Most commercial citrus varieties are susceptible to CBC. However, some citrus varieties and wild citrus germplasms are CBC-resistant and are promising in genetic improvements of citrus resistance against CBC. We aimed to evaluate citrus germplasms for resistance against CBC. First, we developed a rapid evaluation method based on enhanced yellow fluorescent protein (eYFP)-labeled Xcc. The results demonstrated that eYFP does not affect the growth and virulence of Xcc. Xcc-eYFP allows measuring of bacterial titers, but is more efficient and rapid than the plate colony counting method. Next, we evaluated citrus germplasms collected in China. Based on symptoms and bacterial titers, we identified that two citrus germplasms (‘Ichang’ papeda, and ‘Huapi’ kumquat) are resistant, whereas eight citrus germplasms (‘Chongyi’ wild mandarin, ‘Mangshan’ wild mandarin, ‘Ledong’ kumquat, ‘Dali’ citron, ‘Yiliang’ citron, ‘Longyan’ kumquat, ‘Bawang’ kumquat and ‘Daoxian’ wild mandarin) are tolerant. In summary, we have developed a rapid evaluation method to test the resistance of citrus plants against CBC. This method was successfully used to identify two highly canker-resistant citrus germplasms and eight citrus germplasms with canker tolerance. These results could be leveraged in traditional breeding contexts or be used to identify canker resistance genes to improve the disease resistance of commercial citrus varieties via biotechnological approaches.

Effects of Molecular Distillation on the Chemical Components, Cleaning, and Antibacterial Abilities of Four Different Citrus Oils

Essential oils (EOs) from citrus fruits are excellent aromatic resources that are used in food, cosmetics, perfume, and cleaning products. EOs extracted from four citrus varieties, sweet orange, grapefruit, mandarin, and lemon, were separated into two fractions by molecular distillation. The composition, physicochemical properties, cleaning ability, and antimicrobial activity of each EO were then systematically evaluated. The relationships between each of the aforementioned characteristics are also discussed. In keeping with the principle of “like dissolves like,” most citrus EOs show better cleaning ability than acetone and all tend to dissolve the fat-soluble pigment. The key components of citrus EOs are 1-Decanol, α-terpineol, geraniol, and linalool for the inhibition of Staphylococcus aureus, Escherichia coli, Candida albicans, and Vibrio parahaemolyticus, respectively. The findings of this study will be of significant importance for the effective utilization of citrus peel resources and in the development of future applications for citrus EOs.Chemical Compounds Studied in This Article: (+)-α-Pinene (PubChem CID: 6654); β-Phellandrene (PubChem CID: 11142); 3-Carene (PubChem CID: 26049); β-Myrcene (PubChem CID: 31253); D-Limonene (PubChem CID: 440917); γ-Terpinene (PubChem CID: 7461); Octanal (PubChem CID: 454); Decanal (PubChem CID: 8175); Linalool (PubChem CID: 6549); 1-Octanol (PubChem CID: 957); β-Citral (PubChem CID: 643779); α-Terpineol (PubChem CID: 17100); Hedycaryol (PubChem CID: 5365392); α-Citral (PubChem CID: 638011); 1-Decanol (PubChem CID: 8174); Geraniol (PubChem CID: 637566).

Untargeted Metabolomics of Rind Essential Oils Allowed to Differentiate Two Closely Related Clementine Varieties

Chemical characterization of clementine varieties (Citrus clementina Hort. ex Tan.) essential oils (EO) can lead to variety identification and valorization of their potential use in food and aroma industries. The goal of this study was the chemometric discrimination between two very closely related and morphologically identical clementine varieties, Clemenules (NL) and Clemenpons (PO), based on their rind EO, to identify the differential volatile organic compounds (VOCs) and to determine their antioxidant capacity. EO rind volatile profile was determined by gas chromatography coupled to mass spectrometry in Citrus fruit at different ripening stages grown two independent years in two different locations. Untargeted metabolomics and multivariate data analysis showed an evolution of EO volatile profiles markedly parallel in both varieties. Although EO qualitative composition was identical in both varieties, PLS-DA allowed the identification of characteristic VOCs, quantitatively discriminating them along all the ripening process. PO showed higher accumulation of several mono- and sesquiterpene compounds such as trans-carveol, while NL showed higher levels of aldehyde and alcohol non-terpenoids like dodecanal. Both varieties evinced identical EO antioxidant activities, indicating a similar value for food preservation. Hence, untargeted metabolomics approach based on rind EO volatiles was revealed as a powerful technique able to differentiate between morphologically undistinguishable Citrus varieties.

Specific Physiological and Anatomical Traits Associated With Polyploidy and Better Detoxification Processes Contribute to Improved Huanglongbing Tolerance of the Persian Lime Compared With the Mexican Lime

Huanglongbing (HLB) is presently a major threat to the citrus industry. Because of this disease, millions of trees are currently dying worldwide. The putative causal agent is a motile bacteria belonging to Candidatus Liberibacter spp., which is transmitted by psyllids. The bacteria is responsible for the synthesis of callose at the phloem sieve plate, leading to the obstruction of the pores that provide connections between adjacent sieve elements, thus limiting the symplastic transport of the sugars and starches synthesized in leaves to the other plant organs. The Persian triploid lime (Citrus latifolia) is one of the most HLB-tolerant citrus varieties, but the determinants associated with the tolerance are still unknown. HLB-infected diploid Mexican lime (Citrus aurantiifolia) and Persian lime were investigated. The leaf petiole was analyzed using scanning electron microscopy (SEM) to observe callose deposition at the phloem sieve plate. Leaf starch contents and detoxification enzyme activities were investigated. In the field, Persian lime leaves present more limited symptoms due to HLB than the Mexican lime leaves do. Photosynthesis, stomatal conductance, and transpiration decreased compared with control plants, but values remained greater in the Persian than in the Mexican lime. Analysis of the petiole sieve plate in control petiole samples showed that pores were approximately 1.8-fold larger in the Persian than in the Mexican lime. SEM analyses of petiole samples of symptomatic leaves showed the important deposition of callose into pores of Mexican and Persian limes, whereas biochemical analyses revealed better detoxification in Persian limes than in Mexican limes. Moreover, SEM analyses of infected petiole samples of asymptomatic leaves showed much larger callose depositions into the Mexican lime pores than in the Persian lime pores, whereas biochemical traits revealed much better behavior in Persian limes than in Mexican limes. Our results reveal that polyploids present specific behaviors associated with important physiological and biochemical determinants that may explain the better tolerance of the Persian lime against HLB compared with the Mexican lime.

The Return of Asiatic Citrus Canker to Texas: Surveys and Eradication Efforts

Citrus canker caused by Xanthomonas citri subsp. citri (syn. X. axonopodis pv. citri) was first reported in South Texas during the early 1900s, prompting quarantine and eradication programs by the federal government. Its last reported incidence in Texas was from two trees from Corpus Christi in 1943. In May 2016, USDA Animal and Plant Health Inspection Service-Plant Protection and Quarantine (PPQ) confirmed the presence of the Asiatic A strain, which is by far the most widespread and severe form infecting most citrus varieties, on two sour orange trees in a park in Houston. Subsequently, infected trees were detected in a nursery in Richmond (Fort Bend Co.) in August 2016 and in dooryard trees in Pearland (Brazoria Co.) in 2018. PPQ and Texas Department of Agriculture collected a combined 1,949 positive A strain samples from Harris, Fort Bend, and Brazoria Counties during May 2016 to January 2021. These findings resulted in seven active citrus canker quarantine zones being established in the Upper Golf Coast area. The Texas Department of Agriculture is removing positive trees, and surveys are continuing.

Biallelic editing of the LOB1 promoter via CRISPR/Cas9 creates canker-resistant ‘Duncan’ grapefruit

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating citrus diseases worldwide. Generating disease-resistant citrus varieties is considered one of the most efficient and environmentally friendly measures for controlling canker. Xcc causes canker symptoms by inducing the expression of canker susceptibility gene LOB1 via PthA4, a transcription activator-like (TAL) effector, by binding to the effector binding element (EBE) in the promoter region. In previous studies, canker-resistant plants were generated by mutating the coding region or the EBE of LOB1. However, homozygous or biallelic canker-resistant plants have not been generated for commercial citrus varieties, such as grapefruit (C. paradisi), which usually contain two alleles of LOB1 and thus have two types of LOB1 promoter sequences: TI LOBP and TII LOBP. Two different sgRNAs were used to target both EBE types. Both 35S promoter and Yao promoter were used to drive the expression of SpCas9p to modify EBEPthA4-LOBP in grapefruit. Using ‘Duncan’ grapefruit epicotyls as explants, 19 genome-edited grapefruit plants were generated with one biallelic mutant line (#DunYao7). Xcc caused canker symptoms on wild-type and non-biallelic mutant plants but not on #DunYao7. XccPthA4 mutant containing the designer TAL effector dLOB1.5, which recognizes a conserved sequence in both wild-type and #DunYao7, caused canker symptoms on both wild-type and #DunYao7. No off-target mutations were detected in #DunYao7. This study represents the first time that CRISPR-mediated genome editing has been successfully used to generate disease-resistant plants for ‘Duncan’ grapefruit, paving the way for utilizing disease-resistant varieties to control canker.

Damage potential and control of the common mormon butterfly, Papilio polytes Cramer on citrus

Studies were conducted to know the damage potential and effect of insecticides on leaf area feeding and larval mortality of the common Mormon butterfly, Papilio polytes Cramer in the homestead garden and germplasm center of the Patuakhali Science and Technology University (PSTU) campus and in the laboratory, Department of Entomology, PSTU, Patuakhali, Bangladesh during October to December 2016. Ten Citrus host plants such as Kagoji lime-BAU-1, BAU-2, BAU-3, BAU-4, BARI kagoji, elachi lime, sweet orange, orange, jamir and pumelo were used as study materials. To determine the effects of insecticides on leaf area feeding and larval mortality, five treatments viz., T1 = Voliam flexi 300SC @ 0.5 ml/l of water, T2 = Voliam flexi 300SC @ 1.0 ml/l of water T3 = Bioneem Plus @ 0.5 ml/l of water, T4 = Bioneem Plus @ 1.0 ml/l of water T5 = control were applied. Under natural field conditions, the highest percentage of leaf infestation was recorded in orange, followed by BAU-3, BAU-4, malta and elachi lime, while the lowest percentage was in BARI kagoji on 23 and 30 November, 2016. At the top canopy, the highest percentage of leaf infestation was recorded in orange and elachi (56%), while the lowest was in BAU-4 (8%). At the middle canopy, the highest percentage of leaf infestation was recorded in orange (56%), while the lowest was in jambura (12%). At the lower canopy, the highest percentage of leaf infestation was recorded in BAU-3 (40%), while the lowest was in elachi (8%). At the top canopy, significantly the highest percentage of infested leaves per branch was observed in orange (51.39%) and the lowest percentage of infested leaves per branch was in BAU-4 kagoji lime (10.86%). At middle and lower canopies, no significant difference was observed in the percentage of infested leaves per branch among different citrus varieties. The lowest percentage (6%) of leaf area consumed by 4th instar larva was recorded in T2 (Voliam flexi 300SC @ 1.0 ml/l of water) treated leaf, while the highest percentage of leaf area consumption was found in T5 (control) at 3 HAT. The highest percent mortality (100%) of larvae was found in T2 treated citrus plant, while the lowest percent mortality was in the T3 (70%) treated plant. No mortality was recorded in the untreated control (T5) plant. J. Asiat. Soc. Bangladesh, Sci. 47(1): 35-46, June 2021