Citrus Reticulata Cv Research Articles

Investigation of Gut Bacterial Communities of Asian Citrus Psyllid (Diaphorina citri) Reared on Different Host Plants.

Simple SummaryDiaphorina citri is a crucial natural vector of the Huanglongbing pathogen, which has devastated the citrus industry. The host plant is a critical factor that affects insect biology and its symbiont abundance. However, little is known about how host plants affect the bacterial community located in D. citri. In this work, the guts of five different host-plant-feeding populations (i.e., Citrus reticulata cv. Shatangju, Citrus poonensis cv. Ponkan, Murraya paniculata (orange jasmine), Citrus limon (lemon), and Citrus sinensis (navel orange)) were analyzed for bacterial communities by next-generation sequencing. The dominant phylum was Proteobacteria. The most common and abundant bacterial genera in D. citri were Wolbachia, Escherichia-Shigella, and Candidatus Profftella, but their relative abundance varied among the different host plant groups. There were obvious differences in the gut microbiota among the different hosts, and the gut microbe diversity was the highest in the ponkan-feeding population, while the lowest was in the Shatangju-feeding population. Overall, our findings indicate that the host plant can significantly affect the gut microbial community of D. citri. This result can provide new insights into the co-adaptation of D. citri and its symbionts.Diaphorina citri Kuwayama (Hemiptera: Liviidae) can cause severe damage to citrus plants, as it transmits Candidatus Liberibacter spp., a causative agent of Huanglongbing disease. Symbiotic bacteria play vital roles in the ecology and biology of herbivore hosts, thereby affecting host growth and adaptation. In our research, the effects of Rutaceous plants (i.e., Citrus reticulata cv. Shatangju, Citrus poonensis cv. Ponkan, Murraya paniculata (orange jasmine), Citrus limon (lemon), and Citrus sinensis (navel orange)) on the gut microbiota (GM) and microbial diversity of D. citri adults were investigated by 16S rRNA high-throughput sequencing. It was found that Proteobacteria dominated the GM communities. The gut microbe diversity was the highest in the ponkan-feeding population, and the lowest in the Shatangju-feeding population. The NMDS analysis revealed that there were obvious differences in the GM communities among the different hosts. PICRUSt function prediction indicated significant differences in host function, and those pathways were crucial for maintaining population reproduction, growth, development, and adaptation to environmental stress in D. citri. Our study sheds new light on the interactions between symbionts, herbivores, and host plants and expands our knowledge on host adaptation related to GM in D. citri.

Fertilization and pruning improve vegetative growth and architecture of tropical lowland Borneo Prima Mandarin citrus

Vegetative growth of citrus can be increased through a combination of fertilization and pruning. Borneo Prima Mandarin (Citrus reticulata cv Borneo Prima) is a superior local commodity that is grown in lowlands. It has an interesting orange skin, while normally the tropical lowland citrus has a green skin colour. As a new commodity, Borneo Prima Mandarin has to be developed in order to increase production and improve quality. There is no specific cultural practices, so it needs to be developed, especially on fertilizing and pruning. The first experiment aims to observe the plant growth and architecture of plants grown in the orchard. The first factor was the nitrogen fertilization rate (0, 20, 40, 60 g N per tree per application) and the second was pruning (without pruning, open center pruning, hedge pruning). In the second experiment, the plants were applied with the same factors of experiment 1, but were grown on a root observation chamber sized 40 x 20 x 60 cm (length x width x height) which aims to observe the shoot root’s growth and the plant’s biomass. Nitrogen fertilizer had a significant effect to the growth of the Borneo Prima Mandarin, i.e number of shoots and leaves. Twenty grams of nitrogen fertilizer per aplication was efficient to produce new shoots and leaves. Pruning treatments had significant effects towards the plant architecture (i.e reducing plant height, canopy shade projection and canopy length). Open center pruning and hedge pruning made the crown more open which increased the light interception. The shoot grew rapidly two weeks after fertilizing and also pruning. While the root grew rapidly after shoot dormancy.

Aged Pericarpium Citri Reticulatae 'Chachi' Attenuates Oxidative Damage Induced by tert-Butyl Hydroperoxide (t-BHP) in HepG2 Cells.

This study investigated the protective effects of aged Pericarpium Citri Reticulatae ‘Chachi’ (PCR-C) on tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in HepG2 cells. According to HPLC analysis, PCR-C aged 10 years (PCR-C10) had the highest flavonoids content, especially polymethoxyflavones (PMFs), compared with the fresh peel of Citrus reticulata cv. ‘Chachiensis’ and PCR-C aged 1, 3, and 5 years. Then, flavonoids-rich PCR-C samples and non-flavonoids-rich PCR-C samples (NF) were prepared by extracting and purifying PCR-C of different aging periods, for further cell experiments. Pretreatment with flavonoids-rich PCR-C samples (particularly PCR-C10) considerably reversed t-BHP-induced oxidative damage in HepG2 cells by improving cell viability, increasing SOD activity and GSH levels and reducing the overproduction of ROS and MDA. Correlation analysis further indicated that the accumulation of PMFs, mainly 5,6,7,4′-tetramethoxyflavone and nobiletin, was the main reason that PCR-C10 maintained the redox balance in HepG2 cells. These findings provided direct evidence for the cellular antioxidant activity of aged PCR-C and a guide for PCR-C’s classification, authentication and rational use.

First report of post-harvest Fusarium rot of mandarin Citrus reticulata cv. 'Kinnow' caused by Fusarium equiseti in Pakistan.

Citrus reticulata cv. 'Kinnow' mandarin is the most popular and widely grown fruit crop in Pakistan. During 2017, a survey was conducted to the local citrus fruit markets of Faisalabad, Pakistan. Citrus fruits (n=50) exhibiting stem end rot and fruit rot were collected with 15% disease incidence. The stem end region showed light to dark brown lesions and white fungal growth was also observed in the severely infected fruit. Infected fruit were excised into 2mm2 segments, surface disinfected with 1% NaClO, rinsed with sterilized water and dried. Later, these tissues were placed on potato dextrose agar (PDA) medium and subsequently incubated at 25 °C. Purified isolates produced white colonies with beige pigmentation. The frequency of fungal isolation was 47%. Microscopic observations revealed that macroconidia (n=50) had 5 to 6 septations, with a prominent dorsiventral curvature, tapered and elongated apical cell, and a foot shape basal cell. The macroconidia were measuring 22 to 45 × 2.9 to 4.3 µm with an average of 31 × 3.6 µm. However, microconidia were not observed. Chlamydospores were globose, intercalary, solitary, or in pairs, appearing in chains (Leslie and Summerell 2006). For molecular identification, DNA was extracted from all isolates. The internal transcribed spacer region (ITS) ITS1/4 (White et al. 1990), translation elongation factor-1 alpha (TEF) EF1/2 (O'Donnell et al. 1998), and RNA polymerase II subunit 1 (RPB1) (O'Donnell et al. 2013) were amplified using PCR and the product was subsequently sequenced. Based on BLAST analysis, the isolate was identified as Fusarium equiseti (FUS-21). The sequences of the representative isolate FUS-21 were deposited in the GenBank with accession numbers (ITS, MH581300), (TEF, MK203749), and (RPB1, MW596599) showing more than 99% similarity with ITS accession GQ505683, TEF accession GQ505594, and 100% to RPB1 accession JX171481. To determine the pathogenicity, 40 healthy surface disinfested citrus fruit were taken. The fruit were inoculated by creating artificial wounds on the surface with a sterilized needle and 10 μL of 105 spores/mL was deposited in the wounds. In case of control fruit were inoculated with 10 μL sterilized distilled water only, and incubated at 25 °C. All fruit inoculated with the putative pathogen, developed symptoms like the original fruit from which they were isolated. The pathogenicity test was repeated twice. Visible white mycelium appeared at the stem end region and the fruits became dried as the infection progressed. However, the control fruit remained asymptomatic. The pathogen was re-isolated from infected fruit and identified based on morphometric and molecular analysis. Previously we have reported F. oxysporum causing citrus fruit rot in Pakistan (Moosa et al. 2020). This is the first report of F. equiseti causing post-harvest rot of citrus fruits in Pakistan. Kinnow is an important fruit crop of Pakistan with huge export value the management of Fusarium rot is quite important to save the loss of fresh produce.

Comparison of physiochemical characteristics of Citrus reticulata cv. Shatangju fruit with different fruit sizes after storage

‘Shatangju’ (Citrus reticulata) is a loose-skin mandarin cultivar with delicious taste quality. However, its fruit size varied greatly which might affect the storage results. This study investigated the physiochemical characteristics of ‘Shatangju’ fruit with different sizes after a long-term storage, and found that small fruit tended to have a longer shelf life. According to the results, the fruit with a higher transverse diameter tended to contain more seeds and had lower edible rate. The storability of ‘Shatangju’ fruit decreased with the increase of fruit size, in that the big fruit suffered more severe vesicle collapse after storage. Correspondingly, the physiochemical characteristics of the flesh varied among stored fruit of different sizes. The stored larger fruit tended to have higher content of cellulose, hemicellulose and lignin, as well as a lower content of soluble sugars, organic acids, and amino acids in the flesh, which led to the bad texture and tasteless flavor. We also found that the vesicle collapse of ‘Shatangju’ fruit was a very complicated syndrome involving a series of physiochemical changes rather than simple water loss. The results of the present study provided guidance for the classification and storage of ‘Shatangju’ fruit after harvest.

The Effect of Single-Dose Ougan Juice Application on the Pharmacokinetics of Erlotinib.

The aim of our study was to investigate the effects of single-dose Ougan (Citrus reticulata cv. Suavissima) juice application on the pharmacokinetics of erlotinib in vivo. Twelve Sprague-Dawley rats were randomly divided into the Ougan juice and control groups (n = 6 each). The rats were given a single dose of 1 mL/100 g Ougan juice or 1 mL/100 g normal saline (NS) by intragastric administration, followed by a single oral administration of 20 mg/kg erlotinib. The plasma concentration of erlotinib in rats was determined using ultra performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). Erlotinib-d6 was used as the internal standard for chromatographic analysis on the UPLC BEH C18 analysis column (2.1 mm × 50 mm, 1.7 μm). The mobile phase was composed of acetonitrile and 0.1% formic acid eluting by gradient. Different pharmacokinetic (PK) parameters of erlotinib were calculated. The Ougan juice promoted the absorption of erlotinib and reduced the clearance of the drug. The area under the curve of erlotinib in the single-dose Ougan juice pretreatment group was approximately 1.87 times higher, and the maximum blood concentration (Cmax) was approximately 1.34 times higher than that in the control group. The mean residence time of erlotinib in the Ougan juice group was larger, and the clearance rate was smaller than those in the control group; the difference was statistically significant (P < 0.05). Ougan juice affected the PK spectrum of erlotinib in rats by improving the bioavailability of the drug and significantly increasing its plasma concentration.

Extraction process and method validation for bioactive compounds from Citrus reticulata cv. Chachiensis: Application of response surface methodology and HPLC–DAD

AbstractCitrus reticulata cv. Chachiensis, a traditional Chinese herb, has extensive medicinal and edible effects. 3′,4′,5,6,7,8-Hexamethoxyflavone (HM) and 5,6,7,8,4′-pentamethoxyflavone (PM) are main bioactive compounds in Chachiensis, which have been reported to possess various biological properties. In this study, supercritical CO2 extraction (SCE) and high-speed countercurrent chromatography (HSCCC) were utilized to prepare HM and PM from Chachiensis. The contents of target compounds were determined by a high-performance liquid chromatography method with diode-array detection (HPLC-DAD), which was validated using the following parameters: linearity, sensitivity, repeatability, stability, precision and accuracy. The SCE conditions were optimized using response surface methodology with central composite design. Obtained optimum conditions were temperature of 37.9 °C, pressure of 26.3 MPa, and modifier volume of 81.0 mL. Under above conditions, the recoveries of target compounds were 92.52 ± 0.83 and 96.36 ± 0.43%, respectively. The most appropriate solvent system for HSCCC was selected as n-hexane/ethyl acetate/methanol/water (1:0.8:1:1.2, v/v). The HSCCC fractions were detected by HPLC-DAD, liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (1H NMR and 13C NMR). The results indicated that this method was successfully applied to obtain HM and PM with high purities and high recoveries from Chachiensis.

Morphology Characterization, Molecular Phylogeny, and Pathogenicity of Diaporthe passifloricola on Citrus reticulata cv. Nanfengmiju in Jiangxi Province, China.

The Nanfengmiju (Citrus reticulata cv. Nanfengmiju), a high-quality local variety of mandarin, is one of the major fruit crops in Jiangxi Province, China. Citrus melanose and stem-end rot, two common fungal diseases of Nanfengmiju, are both caused by Diaporthe spp. (syn. Phomopsis spp.). Identification of the Diaporthe species is essential for epidemiological studies, quarantine measures, and management of diseases caused by these fungi. Melanose disease was observed on Nanfengmiju fruit in Jiangxi Province of China in 2016. Based on morphological characterization and multi-locus phylogenetic analyses, three out of 39 isolates from diseased samples were identified as D. passifloricola. Since these three isolates did not cause melanose on citrus fruit in the pathogenicity tests, they were presumed to be endophytic fungi present in the diseased tissues. However, our results indicate that D. passifloricola may persist as a symptom-less endophyte in the peel of citrus fruit, yet it may cause stem-end if it invades the stem end during fruit storage. To the best of our knowledge, this is the first report of D. passifloricola as the causal agent of the stem-end rot disease in Citrus reticulata cv. Nanfengmiju.

Identification of Lasiodiplodia pseudotheobromae Causing Fruit Rot of Citrus in China

Considering the huge economic loss caused by postharvest diseases, the identification and prevention of citrus postharvest diseases is vital to the citrus industry. In 2018, 16 decayed citrus fruit from four citrus varieties—Satsuma mandarin (Citrus unshiu), Ponkan (Citrus reticulata Blanco cv. Ponkan), Nanfeng mandarin (Citrus reticulata cv. nanfengmiju), and Sugar orange (Citrus reticulata Blanco)—showing soft rot and sogginess on their surfaces and covered with white mycelia were collected from storage rooms in seven provinces. The pathogens were isolated and the pathogenicity of the isolates was tested. The fungal strains were identified as Lasiodiplodia pseudotheobromae based on their morphological characteristics and phylogenetic analyses using the internal transcribed spacer regions (ITS), translation elongation factor 1-α gene (TEF), and beta-tubulin (TUB) gene sequences. The strains could infect wounded citrus fruit and cause decay within two days post inoculation, but could not infect unwounded fruit. To our knowledge, this is the first report of citrus fruit decay caused by L. pseudotheobromae in China.

Reduced expression of CsPH8, a P-type ATPase gene, is the major factor leading to the low citrate accumulation in citrus leaves

Citrate is an important intermediate product for the biosynthesis of several metabolites in plants. As two important organs of the citrus plant, fruits and leaves have their own metabolites characteristics; among them, citrate is normally high in fruit juice sacs (JS) and low in leaves. In this study, citrate content and transcript levels of citrate synthesis, transport, storage, and utilization related genes were compared between leaves and fruit JS of Citrus reticulata cv. ‘Huagan No. 2’, C. grandis cv. ‘Hirado Buntan’, and C. sinensis cv. ‘Anliu’. Results indicated that the citrate content in fruit JS was significantly higher than in leaves of each cultivar. Only the relative mRNA levels of a P-type proton pump gene, CsPH8, was significantly lower in leaves than in fruit JS of three citrus cultivars, while other genes related to citrate biosynthesis, transport, storage, and utilization were highly expressed in leaves as compared to fruit JS. Furthermore, CsPH8 transient and stable transformation in leaves indicated that the change in citrate content is highly consistent with the change of CsPH8 transcript levels. Taken together, our results strongly suggest that the low accumulation of citrate in citrus leaves is mainly due to the low expression level of CsPH8; additionally, the high level of expression of citrate-utilizing genes would prevent citrate accumulation in the leaf organ.

Inhibitory effects of fermented Ougan (Citrus reticulata cv. Suavissima) juice on high-fat diet-induced obesity associated with white adipose tissue browning and gut microbiota modulation in mice.

In this study, Ougan juice (OJ) and lactic acid bacteria fermented Ougan juice (FOJ) were investigated individually for their capability of preventing obesity in high-fat diet (HFD)-fed C57BL/6J mice. After being administered with OJ or FOJ for 10 weeks, the body weight gain, hyperlipidemia, and gut microbiota dysbiosis of HFD-fed mice were examined. The results showed that OJ or FOJ supplementation inhibited weight gain, lowered fat accumulation, reduced liver steatosis, improved glucose homeostasis and insulin sensitivity, increased brown adipose tissue (BAT) activity, and promoted white adipose tissue (WAT) browning. Both OJ and FOJ additions increased the diversity of gut microbiota. OJ reduced the relative abundance of phylum Erysipelatoclostridiaceae and genus Erysipelatoclostridium and remarkably increased SCFA-producing bacteria Blautia, while FOJ reduced the ratio of Firmicutes to Bacteroidetes and enhanced the relative abundance of family Lactobacillaceae. Spearman's correlation analysis revealed that Akkermansia, Dubosiella, and Muribaculaceae were significantly negatively correlated with obesity-related indexes. In general, FOJ exhibited a better inhibitory effect on obesity than OJ, and the possible inhibitory mechanism lies in promoting WAT browning and increasing intestinal probiotics. This study provides the guidance for developing fermented Ougan juice as an obesity-inhibiting functional food.

Comparative analysis of bacterial and fungal endophytes responses to Candidatus Liberibacter asiaticus infection in leaf midribs of Citrus reticulata cv. Shatangju

The endophytic bacterial and fungal profiles were comparatively studied between Citrus Huanglongbing (HLB) infected and uninfected healthy leaves of a mandarin cultivar, Shatangju (Citrus reticulata Blanco), by using the next generation sequencing technology. A total of 53 endophytic bacterial orders in12 phyla and 24 endophytic fungal orders in 2 phyla were detected in healthy, asymptomatic and symptomatic leaf midribs. It was found that the predominant endophytic bacteria belonged to Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes and the predominant endophytic fungi belonged to Ascomycota and Basidiomycota. Operational taxonomic unit (OTU) analysis showed that OTU_2 and OTU_6 were the core endophytic fungal OTUs, and OTU_10, OTU_4 and OTU_141 were the core endophytic bacterial OTUs. The richness and diversity of the endophytic microbes were affected by infection of the HLB bacterium, Candidatus Liberibacter asiaticus (CLas). The most significant change was the reduction in endophytic fungal OTUs following CLas infection. Eleven endophytic fungal OTUs and 24 endophytic bacterial OTUs were found to be associated with tree healthy status in citrus. Some of the healthy and HLB disease specific OTUs were discussed, with emphasis on their possible roles in HLB pathogenesis.

Characterization of antioxidant compounds extracted from Citrus reticulata cv. Chachiensis using UPLC-Q-TOF-MS/MS, FT-IR and scanning electron microscope

Citrus reticulata cv. Chachiensis (CRC) has been widely used in many Chinese medicine prescriptions and developed as various health products. Due to its extensive biological activities, increased interest has focused on CRC as a potential source for the treatment of various diseases. However, there is no systematic and sufficient research on optimization and characterization of ultrasound extraction of antioxidant compounds from CRC up to now. In the present work, three parameters including ultrasonic amplitude (45, 55, 70, 85, 95 %), ultrasonic time (26, 40, 60, 80, 94 min) and ethanol concentration (16, 30, 50, 70, 84 %, v/v) were estimated using central-composite design. Determined optimum conditions, considering four responses (TPC, TFC, DPPH and ABTS) at the same time, were 72 % amplitude, 80 min and 70 % ethanol concentration. The maximum yields were TPC values (145.73 mg GAE/g), TFC values (135.21 mg QE/g), DPPH and ABTS scavenging capacity (87.88 and 76.18 mg TE/g, respectively). 55 phytochemicals were identified in the CRC extract using the UPLC-QTOF-MS/MS, 34 of which were flavonoids, which provided important support for various potential applications of CRC.

Polymethoxyflavones from citrus inhibited gastric cancer cell proliferation through inducing apoptosis by upregulating RARβ, both in vitro and in vivo

In order to discover the active anti-tumor ingredients during the flavonoids separation process of Ougan (Citrus reticulata cv. Suavissima), gastric cancer cell lines including AGS, BGC-823, and SGC-7901 were employed to evaluate the proliferation inhibition abilities of Ougan extracts, flavanone components, polymethoxyflavone components, neohesperidin, nobiletin, tangeretin, and 5-demethylnobiletin. Quantitative real-time PCR was used to detect the expression of three retinoic acid receptor genes, including RARA, RARB, and RARG. Western blot and immunohistochemistry were used to detect protein expressions. The results showed that the polymethoxyflavone components and the PMFs monomers inhibited the proliferation of three gastric cancer cell lines and induced apoptosis. The mechanism exploration found that PMFs up-regulated the expression of the RARB gene selectively and activated the Caspase3, 9, and PARP1 proteins. In addition to 5-demethylnobiletin, other PMFs also upregulated the expression of cleaved Caspase8. The mechanism was preliminarily verified by a RARβ inhibitor AGN 193109. Moreover, a nude mice tumor xenograft model confirmed the tangeretin could exhibit in vivo anti-tumor effect through inducing apoptosis and upregulating RARβ protein. All result suggested that tangeretin may be a potentially novel, safe and effective drugs with less toxicity and lesser side effects for gastric cancer therapeutics.

Beneficial Regulatory Effects of Polymethoxyflavone-Rich Fraction from Ougan (Citrus reticulata cv. Suavissima) Fruit on Gut Microbiota and Identification of Its Intestinal Metabolites in Mice.

Polymethoxyflavones (PMFs) are special flavonoids in citrus fruits that have been suggested to be beneficial to human health. However, whether PMFs in citrus fruit alter human gut microbiota is not well understood. The aim of the present study was to investigate the effects of PMF-rich fraction from Ougan (Citrus reticulata cv. Suavissima) on gut microbiota and evaluate the intestinal metabolic profile of PMFs in Institute of Cancer Research mice. The main components of the PMF-rich fraction were nobiletin, tangeretin, and 5-demethylnobiletin. The composition of the gut microbiota was analyzed using 16S ribosomal DNA sequencing. The results showed that after oral administration, the composition of mice gut microbiota was significantly altered. The relative abundance of two probiotics, Lactobacillus and Bifidobacterium, were found to increase significantly. A total of 21 metabolites of PMFs were detected in mice intestinal content by high performance liquid chromatography electrospray ionization tandem mass spectrometry, and they were generated through demethylation, demethoxylation, hydroxylation, and glucuronidation. Our results provided evidence that PMFs have potential beneficial regulatory effects on gut microbiota that in turn metabolize PMFs, which warrants further investigation in human clinical trials.

A NAC transcription factor and its interaction protein hinder abscisic acid biosynthesis by synergistically repressing NCED5 in Citrus reticulata.

Although abscisic acid (ABA) is a vital regulator of fruit ripening and several transcription factors have been reported to regulate ABA biosynthesis, reports of the effect of ABA on citrus ripening and the regulation of its biosynthesis by a multiple-transcription-factor complex are scarce. In the present study, a systematic metabolic, cytological, and transcriptome analysis of an ABA-deficient mutant (MT) of Citrus reticulata cv. Suavissima confirmed the positive effect of ABA on the citrus ripening process. The analysis of transcriptome profiles indicated that CrNAC036 played an important role in the ABA deficiency of the mutant, most likely due to an effect on the expression of 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5). Electrophoretic mobility shift assays and dual luciferase assays demonstrated that CrNAC036 can directly bind and negatively regulate CrNCED5 expression. Furthermore, yeast two-hybrid, bimolecular fluorescence complementation, and dual luciferase assays demonstrated that CrNAC036 interacted with CrMYB68, also down-regulating the expression of CrNCED5. Taken together, our results suggest that CrNAC036 and CrMYB68 synergistically inhibit ABA biosynthesis in citrus fruit by regulating the expression of CrNCED5.

Distinct microbial communities among different tissues of citrus tree Citrus reticulata cv. Chachiensis

Plant microbiota colonize all organs of a plant and play crucial roles including supplying nutrients to plants, stimulating seed germination, promoting plant growth, and defending plants against biotic and abiotic stress. Because of the economic importance, interactions between citrus and microbes have been studied relatively extensively, especially citrus-pathogen interactions. However, the spatial distribution of microbial taxa in citrus trees remains under-studied. In this study, Citrus reticulata cv. Chachiensis was examined for the spatial distribution of microbes by sequencing 16S rRNA genes. More than 2.5 million sequences were obtained from 60 samples collected from soil, roots, leaves, and phloem. The dominant microbial phyla from all samples were Proteobacteria, Actinobacteria and Acidobacteria. The composition and structure of microbial communities in different samples were analyzed by PCoA, CAP, Anosim and MRPP methods. Variation in microbial species between samples were analyzed and the indicator microbes of each sample group were identified. Our results suggested that the microbial communities from different tissues varied significantly and the microenvironments of tree tissues could affect the composition of its microbial community.

Physiochemical changes in Citrus reticulata cv. Shatangju fruit during vesicle collapse

‘Shatangju’ (Citrus reticulate) is a popular mandarin that easily develop vesicle drying, which is one of the most common internal physiological disorders of citrus fruit. The vesicle drying occurred firstly in the peel-side of the segment of ‘Shatangju’ fruit, and then the juice sac shrank gradually with the development of the syndrome, which finally resulted in the collapse and hollowing of the segments. The incidence of vesicle collapse increased with the storage temperature and the seed number. After storage at 10 °C and 20 °C for 90 d, over 80 % of the fruit developed vesicle collapse syndromes. The physiochemical characteristics of healthy segments and the vesicle collapsed segments were subsequently compared in detail. The results showed that, the physiochemical changes of ‘Shatangju’ fruit during vesicle collapse were characterized by the increase of H2O2, MDA, flavonoids and cell wall compounds, and by the decrease of carotenoids, ethanol, ethyl acetate, d-limonene and β-myrcene, as well as by the modest decrease of sugars and acids. In addition, the contents of amino acids in the vesicle collapsed segment changed slightly. Taken together, the results indicated that the vesicle collapse of ‘Shatangju’ fruit involved in the changes of primary metabolism, secondary metabolism as well as the peroxidation of the tissue. Present study provided detailed information about the physiochemical alterations occurring in the vesicle collapsed segment of ‘Shatangju’ mandarin.

Metabolomics Analysis of the Peels of Different Colored Citrus Fruits (Citrus reticulata cv. 'Shatangju') During the Maturation Period Based on UHPLC-QQQ-MS.

Citrus is a globally consumed fruit with great popularity. Mandarin (Citrus reticulata cv. ‘Shatangju’) is a local variety, and its planting area and yield are the greatest regarding fruit tree planting in Guangdong Province, China. However, its resistance to Huanglongbing (HLB) is weak. After infection by HLB, the fruits cannot develop normally. In this study, four kinds of fruits were classified as HBG, XQG, ZQG, and DHG, according to the color of their peels. The metabolomes of the three abnormally colored groups (HBG, XQG, and ZQG) and the normally colored group (DHG) were compared using a UPLC-QQQ-MS-based metabolomics approach. In total, 913 metabolites were identified and classified into 23 different categories, including phenylpropanoids and flavonoids; among them, 215 (HBG, 177; XQG, 124; and ZQG, 62) metabolites showed differential accumulation in the three comparison groups (HBG/XQG/ZQG versus DHG). A total of 2 unique metabolites, O-caffeoyl maltotriose and myricetin were detected only in DHG samples. When comparing HBG with DHG, there were 109 decreased and 68 increased metabolites; comparing XQG with DHG, there were 88 decreased and 36 increased metabolites; comparing ZQG with DHG, 41 metabolites were decreased, and 21 metabolites were increased. Metabolic pathway enrichment analysis of these differential metabolites showed significant enrichment of the “phenylpropanoid biosynthesis” pathway in all comparison groups. The hierarchical cluster analysis of the differential metabolites of the four groups showed a clear grouping patterns. The relative contents of three phenylpropanoids, four flavonoids, two alkaloids, one anthocyanin, and two other metabolites were significantly different between each comparison group. This study might provide fundamental insight for the isolation and identification of functional compounds from the peels of citrus fruit infected with HLB and for in-depth research on the effect of HLB on the formation of fruits pigment and the development of HLB-resistant citrus varieties.

Distribution and incidence of Huanglongbing in Citrus reticulata cv. Kinnow mandarin in Punjab

Huanglongbing (HLB)/citrus greening is destroying millions of citrus trees globally including India. HLB is caused by phloem limited, non-culturable alpha-proteobacteria, under genus Candidatus Liberibacter (Ca. L) which predominantly vectored by psyllid (Diaphorina citri). The PCR-based technique was used for detection of Ca. L asiaticus (Ca. Las) associated with HLB targeting partial 16S rRNA. No recent report on incidence and distribution of HLB in Kinnow mandarin (Citrus reticulata cv. Kinnow) orchards of Punjab of India is available. Based on extensive survey and PCR, incidence and distribution of HLB in Punjab was determined. High HLB incidence of 17.5–39.1% in Punjab was observed. Occurrence of psyllids in mandarin orchards of Ludhiana in Punjab was observed. The PCR based diagnostic method used in this study is suitable for a rapid detection of Ca. Las in infected mandarin trees in India.