Distribution and genetic diversity of Coguvirus eburi in South African citrus and the development of a real-time RT-PCR assay for citrus-infecting coguviruses

Citrus virus A (CiVA), a novel negative-sense single-stranded RNA virus assigned to the species Coguvirus eburi in the genus Coguvirus, was detected in South Africa with the use of high-throughput sequencing (HTS) after its initial discovery in Italy. CiVA is closely related to citrus concave gum-associated virus (CCGaV), recently assigned to the species Citrus coguvirus. Disease association with CiVA is however incomplete. CiVA was detected in grapefruit (Citrus paradisi Macf.), sweet orange (C. sinensis (L.) Osb.) and clementine (C. reticulata Blanco) in South Africa and a survey to determine the distribution, symptom association and genetic diversity was conducted in three provinces and seven citrus production regions. The virus was detected in ‘Delta’ Valencia trees in six citrus production regions and a fruit rind symptom was often observed on CiVA-positive trees. Additionally, grapefruit showing symptoms of citrus impietratura disease were positive for CiVA. This virus was primarily detected in older orchards that were established prior to the application of shoot tip grafting for virus elimination in the South African Citrus Improvement Scheme. The three viral encoded genes of CiVA isolates from each cultivar and region were sequenced to investigate sequence diversity. Genetic differences were detected between the ‘Delta’ Valencia, grapefruit and clementine samples, with greater sequence variation observed with the nucleocapsid protein (NP) compared to the RNA-dependent RNA polymerase (RdRp) and the movement protein (MP). A real-time detection assay, targeting the RdRp, was developed to simultaneously detect citrus infecting coguviruses, CiVA and CCGaV, using a dual priming reverse primer to improve PCR specificity.

Enumeration of Citrus endophytic bacterial communities based on illumine metagenomics technique

Citrus is a valuable crop in Pakistan because it is rich in vitamin C and antioxidants. Huanglongbing (HLB) has an influence on citrus production around the world caused by a bacterium “ Candidatus liberibacter asiaticus ” (CLas), africanus and americanus. The structure and diversity of bacterial species in various ecosystems can be quickly examined using NGS. This approach is considerably quicker and more precise than outdated methods. Healthy or citrus greening infected leaf samples of Grapefruit, Citrus aurantifolia , and Citrus reticulata Blanco was used for diversity analysis. In this study high throughput, NGS technique was used to access the population of both cultivable and non-cultivable bacterial endophytes from citrus leaves, by using PCR amplicons of 16S rDNA sequences (V5–V7 regions) with Illumina Hi seq. As a result, a total number of 68,722 sequences were produced from the test samples. According to the NGS-based diversity classification, the most common genera of exploited bacterial endophytes were Proteobacteria, Firmicutes, Bacteroides, Cyanobacteria, and Actinobacteria. Citrus aurantifolia and Citrus paradisi showed almost equal diversity, whereas Citrus reticulata Blanco had a higher proportion of Proteobacteria and Cyanobacteria in their leaves. To determine alpha diversity (AD), additional data was analyzed using statistical indices such as Shannon, Chao1, and Simpson. According to the inverse Simpson diversity index, the abundance of the microbial population in six different citrus samples was 0.48, 0.567, and 0.163, respectively. The metagenomics of microbiota in plant tissues was successfully recorded by NGS technology, which can help us learn more about the interactions between plants and microbes. This research is the first step toward a better understanding of 16SrRNA-based metagenomics from citrus in Pakistan using Illumina (Hi seq) Technology.

Anti-plasmodial Effect of C. limon and C. paradisi extracts on Plasmodium berghei-infected mice

Antiplasmodial effect of Citrus limon and Citrus paradisi extracts on Plasmodium berghei-infected mice was studied. Twenty five albino mice were randomized into five categories of G, L, GL, ACT (positive control) and NC which stand for grape, lemon, grape and lemon combined extracts, artemisinin combined therapy and negative control respectively. The NC group did not receive any intervention. Other treatments were administered orally for 12 days whereas administration of ACT lasted for 3 days. Blood was collected from the tail vein of the mice at a three day interval through venipuncture. Thick blood films were prepared and parasite densities were estimated using standard parasitological techniques. Results were analysed with ANOVA and Duncan multiple range tests. There was no significant difference (p>0.05) between parasite densities of the treatment groups and the negative control at baseline levels. However, as the treatment progressed from day 3 through day 9, there were significant reductions (p<0.05) in parasite densities among treatment groups when compared to the negative control. In this study, extracts of C. limon and C. paradisi in both single and combined strengths have been found to have antiplasmodial properties in mice. ACT possessed the highest antiplasmodial effect while C. limon as a single treatment ranked second in possession of antiplasmodial activity but exhibited increased RBC lysis. In combination, C. limoni and C. paradise extracts showed antiplasmodial activity that is slightly less than that exhibited by the lemon extract alone, but maintained normal RBC morphology whereas C. paradisi extract alone exhibited the lowest level of parasite clearance with atrophied red blood cells. Investigation of the effects of the extracts on liver, kidney and gastrointestinal tissues of mice is recommended before they could be prescribed as antimalaria for other animals and humans.

Distribution of minerals between orange juice and orange flesh in various cultivars

The aim of this study was to evaluate the distribution of essential and toxic elements between the orange juice and orange flesh of various orange cultivars. Different orange cultivars, such as Abousorah [Citrus sinensis (L.)], Aseear (Citrus aurantium), Afandi (Citrus reticulata Blanco), Helo (Citrus sinensis), and grapefruit (Citrus paradisi), were collected from local markets. Elemental analysis was carried out after microwave-assisted digestion using inductively coupled plasma mass spectrometry (ICP-MS) in 18 samples. Eleven elements (V, Cr, Mn, Co, Ni, Cu, Zn, As, Se, Cd, and Pb) were analysed. Their average concentrations ranged from 0.006 µg g^–1 to 7.13 µg g^–1 in both orange juice and flesh (wet weight). There was a high increase in the average concentrations of all elements in the juice compared to the flesh of up to 40-fold.

Regulation of Tocopherol Biosynthesis During Fruit Maturation of Different Citrus Species

Tocopherols are plant-derived isoprenoids with vitamin E activity, which are involved in diverse physiological processes in plants. Although their biosynthesis has been extensively investigated in model plants, their synthesis in important fruit crops as Citrus has scarcely been studied. Therefore, the aim of this work was to initiate a physiological and molecular characterization of tocopherol synthesis and accumulation in Citrus fruits during maturation. For that purpose, we selected fruit of the four main commercial species: grapefruit (Citrus paradisi), lemon (Citrus limon), sweet orange (Citrus sinensis), and mandarin (Citrus clementina), and analyzed tocopherol content and the expression profile of 14 genes involved in tocopherol synthesis during fruit maturation in both the flavedo and pulp. The selected genes covered the pathways supplying the tocopherol precursors homogentisate (HGA) (TAT1 and HPPD) and phytyl pyrophosphate (PPP) (VTE5, VTE6, DXS1 and 2, GGPPS1 and 6, and GGDR) and the tocopherol-core pathway (VTE2, VTE3a, VTE3b, VTE1, and VTE4). Tocopherols accumulated mainly as α- and γ-tocopherol, and α-tocopherol was the predominant form in both tissues. Moreover, differences were detected between tissues, among maturation stages and genotypes. Contents were higher in the flavedo than in the pulp during maturation, and while they increased in the flavedo they decreased or were maintained in the pulp. Among genotypes, mature fruit of lemon accumulated the highest tocopherol content in both the flavedo and the pulp, whereas mandarin fruit accumulated the lowest concentrations, and grapefruit and orange had intermediate levels. Higher concentrations in the flavedo were associated with a higher expression of all the genes evaluated, and different genes are suitable candidates to explain the temporal changes in each tissue: (1) in the flavedo, the increase in tocopherols was concomitant with the up-regulation of TAT1 and VTE4, involved in the supply of HGA and the shift of γ- into α-tocopherol, respectively; and (2) in the pulp, changes paralleled the expression of VTE6, DXS2, and GGDR, which regulate PPP availability. Also, certain genes (i.e., VTE6, DXS2, and GGDR) were co-regulated and shared a similar pattern during maturation in both tissues, suggesting they are developmentally modulated.