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 This approach is considerably quicker and more precise than outdated methods.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.

Hesperidin Extracted from Citrus reticulata Blanco Protects Cardiac Mitochondria Against Hypoxia/Reoxygenation Injury

This study was conducted to evaluate the protective effect of Hesperdin (Hes) extracted from Citrus reticulata Blanco on cardiac mitochondria in hypoxia/reoxygenation (HR) injury in vitro. Methods: H9C2 cardiomyocytes were cultured under normal (control), HR, and treatment conditions. The reactive oxygen species and calcium levels in experimental groups were analyzed by using suitable fluorescence kits. Results: The obtained results showed that the addition of Hes at dose of 0,01562 mg/mL sharply decreased the mitochondrial oxidative stress of H9C2 cells under HR conditions. In particular, Hes showed the remarkable efficiency in maintaing cellular calcium levels. In HR-exposed H9C2 cell group, the hydrogen peroxide and superoxide levels were highly increased compared to those in control group (1,54±0,06 and 1,74±0,38, p<0,05). HR also strongly induced the elevation of cytosolic Ca²⁺ and mitochondial Ca²⁺ of H9C2 cardiomyocytes with the values were 1,96±0,05% and 1,62±0,33 (ratio to control, p<0,05), respectively. Interestingly, post-hypoxic supplementation of Hes effectivelly abolished the negative incresement of these indicators with the lower levels of reactive oxygen species and the better modulation of Ca²⁺ homeostasis. Conclusion: The present results are pilot data on the effects of Hes in protecting cardiac mitochondria against HR injury.

Rapid Discrimination of Citrus reticulata ‘Chachi’ by Electrospray Ionization–Ion Mobility–High-Resolution Mass Spectrometry

A common idea is that some dishonest businessmen often disguise Citrus reticulata Blanco varieties as Citrus reticulata ‘Chachi’, which places consumers at risk of economic losses. In this work, we combined high-resolution ion mobility (U-shaped mobility analyzer) with high-resolution mass spectrometry to rapidly distinguish Citrus reticulata ‘Chachi’ from other Citrus species. The samples were analyzed directly through simple extraction and the analytes were separated in one second. It only took about 1 min to perform a cycle of sample analysis and data acquisition. The results showed that polymethoxylated flavones and their isomers were separated easily by the ion mobility analyzer and preliminarily identified according to the accurate mass. Moreover, the collision cross-section values of all analytes, which could be used as auxiliary parameters to characterize and identify the compounds in the samples, were measured. Twenty-four samples were grouped as two clusters by multivariate analysis, which meant that Citrus reticulata ‘Chachi’ could be effectively differentiated. It was confirmed that the developed method had the potential to rapidly separate polymethoxylated flavones and distinguish between Citrus reticulata ‘Chachi’ and other Citrus reticulata Blanco varieties.

Preventive Effect of Gonggan (Citrus Reticulata Blanco Var. Gonggan) Peel Extract on Ethanol/HCl-Induced Gastric Injury in Mice via an Anti-oxidative Mechanism

Gonggan (Citrus reticulata Blanco var. gonggan) is one of the most popular citruses. In this study, the effect of Gonggan peel extract (GPE) on gastric injury was investigated. The components in GPE were analysed by HPLC and the gastric injury model in mice was established by ethanol/hydrochloric acid. After treatment by GPE, the pathological changes of gastric tissue were observed by optical microscope. The levels of oxidative stress and inflammation were measure by kit. And the mRNA expression of related gene was determined by qPCR assay. HPLC result showed GPE mainly contained the flavonoids narirutin, hesperidin, nobiletin, tangeretin and 5-demethylnobiletin. Morphological and pathological analysis of gastric tissue revealed that GPE could relieve gastric injury. Also, GPE increased the levels of SOD, GSH-Px, and CAT and decreased the level of MDA. Moreover, GPE decreased the levels of the inflammatory cytokines TNF-α, IFN-γ, IL-1β, and IL-6 to suppress inflammation. In addition, the q-PCR results showed that GPE upregulated the mRNA expression of SOD1, SOD2, γ-GCS, GSH-Px, CAT, and IκBα and downregulated the mRNA expression of NF-κB. In conclusion, GPE alleviated gastric injury caused by ethanol/hydrochloric acid by inhibiting oxidative stress and the inflammatory response. The mechanism by which GPE protects gastric tissues may involve the antioxidative pathway. Therefore, GPE has great potential to be developed as a product to prevent gastric injury.