Quantitative real-time reverse transcription polymerase chain reaction (qPCR) has become the preferred method for studying low-abundant mRNA expression. Appropriate application of qPCR in such studies requires the use of reference gene(s) as an internal control in order to normalize the mRNA levels between different samples for an exact comparison of gene expression levels. Expression of the reference gene should be independent from development stage, cell/tissue types, treatments and environmental conditions. Recognizing the importance of reference gene(s) in normalization of qPCR data, various reference genes have been evaluated for stable expression under specific conditions in various organisms. In plants, only a few of them have been investigated, and very few reports about such reference genes in citrus. In the present study, seven candidate reference genes (18SrRNA, ACTB, rpII, UBQI, UBQ10, GAPDH and TUB) were tested, and three of them (18SrRNA, ACTB and rpII) proved to be the most stable ones among six leaf samples of different citrus genotypes. The three candidate reference genes were further analyzed for their stability of expression in five different tissues, and the results indicated that they were not completely stable. It is commonly accepted that gene expression studies should be normalized using more than one reference gene. Based on our results, we propose the use of the mean result rendered by18SrRNA, ACTB and rpII as reference genes to normalize mRNA levels in qPCR analysis of diverse cultivars and tissues of citrus. These results may provide a guideline for future works on gene expression in citrus by using qPCR.

The COOH terminal of pthA encoding three nuclear localizing signals (NLS) was amplified by polymerase chain reaction (PCR) from the plasmid of Xanthomonas axonopodis pv. citri, the pathogen of citrus canker disease. Then the sense and antisense strands of the nls were cloned into pBI121 vector. pthA-nls driven by the CaMV35 s promoter was transferred into sweet orange via Agrobacterium -mediated transformation. Successful integration was confirmed by PCR and Southern blotting, and 12 sense-nls (nls (+)) and 9 antisense-nls (nls (-)) transgenic clones were obtained. The expression of nls fragment was analyzed by RT-PCR, Real time q-PCR and Western blotting, in which the specific NLS protein was detected only in nls (+) transgenic clones. In an in vitro assay, when pin-puncture inoculation was performed with 2.5 × 10(7) cfu/ml of bacterial solution, the nls (+) transgenic clones showed no typical lesion development, while typical symptoms were observed in the wild types and the nls (-) transgenic clones. In vivo assay results indicated that the nls (+) transgenic clones showed less disease incidence, in comparison with the wild types and the nls (-) transgenic clones, when pin-puncture inoculation was performed with 10(4)-10(5) cfu/ml. The minimum disease incidence was 23.3% for 'Sucarri' sweet orange and 33.3% for 'Bingtang' sweet orange. When 10(4)-10(7) cfu/ml of pathogen was spray inoculated, the nls (+) transgenic clones did not show any symptom, and even the concentration raised to 10(9) cfu/ml, the disease incidence was 20-80%, while the wild types and the nls (-) transgenic clones had 100% disease development with whatever concentration of inoculum. Two transgenic clones were confirmed to be resistant to citrus canker disease in the repeated inoculation. The results suggested that the transformation of nls sense strands may offer an effective way to acquire resistance to citrus canker disease.

Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus bacterial canker, an economically important disease to world citrus industry. To monitor the infection process of Xac in different citrus plants, the enhanced green florescent protein (EGFP) visualizing system was constructed to visualize the propagation and localization in planta. First, the wild-type Xac was isolated from the diseased leaves of susceptible 'Bingtang' sweet orange, and then the isolated Xac was labeled with EGFP by triparental mating. After PCR identification, the growth kinetics and pathogenicity of the transformants were analyzed in comparison with the wild-type Xac. The EGFP-labeled bacteria were inoculated by spraying on the surface and infiltration in the mesophyll of 'Bingtang' sweet orange leaves. The bacterial cell multiplication and diffusion processes were observed directly under confocal laser scanning microscope at different intervals after inoculation. The results indicated that the EGFP-labeled Xac releasing clear green fluorescence light under fluorescent microscope showed the infection process and had the same pathogenicity as the wild type to citrus. Consequently, the labeled Xac demonstrated the ability as an efficient tool to monitor the pathogen infection.

Abstract:The objectives of the current study were to isolate and identify the pathogen responsible for citrus canker and investigate the efficacy of sulfone derivatives containing 1,3,4-oxadiazole moiety on controlling citrus canker caused by Xanthomonas citri subsp. citri (Xcc) under in vitro and field conditions. In an in vitro study, we tested eight sulfone derivatives against Xcc and the results demonstrated that compound 3 exhibited the best antibacterial activity against Xcc, with a half-maximal effective concentration (EC50) value of 1.23 μg/mL, which was even better than those of commercial bactericides Kocide 3000 (58.21 μg/mL) and Thiodiazole copper (77.04 μg/mL), respectively. Meanwhile, under field experiments, compound 3 treatments demonstrated the highest ability to reduce the disease of citrus canker in leaves and fruits in two different places relative to an untreated control as well as the commercial bactericides Kocide 3000 and Thiodiazole copper. Meanwhile, compound 3 could stimulate the increase in peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia lyase (PAL) activities in the navel orange leaves, causing marked enhancement of plant resistance against citrus canker. Moreover, compound 3 could damage the cell membranes, destruct the biofilm formation, inhibit the production of extracellular OPEN ACCESSMolecules 2015, 20 14104 polysaccharide (EPS), and affect the cell membrane permeability to restrain the growth of the bacteria.

In order to provide a theoretical basis for conservation and utilization of excellent flowering cherry germplasms, genetic diversity and genotype were analyzed based on single nucleotide polymorphism (SNP) molecular markers. A total of 47 germplasms were sequenced using restriction site-associated DNA sequencing (RAD-Seq) method, and highly consistent SNP sites have been identified for genetic diversity analysis and genotyping. The RAxML program was used for phylogenetic tree construction, based on the maximum likelihood (ML) method. The ADMIXTURE software was used to analyze genetic structure. The GCTA software was used for principal component analysis. Genetic diversity parameters, includes nucleotide diversity (π value), expected heterozygosity (exp He) and genetic differentiation index (FST), were carried out using the PopGenome and Arlequin 3.0 software. A total of 79 667 highly consistent SNPs were obtained, after genotype integrity filtering and closely linked sites screening. According to the phylogenetic, principal components and population structure analysis results, the 47 germplasms could be divided into eight genetic clusters. The π and exp He value of each cluster was as follows:

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Prunus subgenus Cerasus is a large and diverse genus. Its botanical classification has long been controversial and complicated. Molecular markers derived from the chloroplast genome may provide useful tools for phylogenetic resolution and taxonomic study in Prunus species. Thus, we compared chloroplast genomes of 23 Prunus species to identify sequences with high variation levels. The repeated sequences (RSs) and two types of sequence variation, comprising divergent homologous regions and short sequence repeats (SSRs), were identified. The species with the highest number of RSs was P. emarginata (60), whereas P. campanulata contained the highest amount of SSRs (70). In contrast, P. dictyoneura and P. yedoensis contained the lowest number of RSs (37) and SSRs (46), respectively. Out of these homologous SSRs, 11 were shared in the 23 species, and seven of them showed variations. A total of 203 homologous regions were identified. Out of these, 20 regions (19 IGSs and one intron) showed a high variation from 6.75 to 19.31%. The phylogenetic tree which was inferred based on 19 highly variable regions showed a topology similar to the one obtained using the whole chloroplast genome. We conclude that the seven variable SSRs and the 20 highly variable regions may potentially be convenient as molecular markers for phylogenetic and population genetics studies in subgenus Cerasus species. In addition, the molecular phylogenetic position of P. clarofolia was inferred based on the 19 highly variable regions.

Prunus conradinae is a flowering cherry species with high ornamental value. In this study, the complete chloroplast (cp) genome of P. conradinae was obtained using a genome skimming approach. The cp genome was 158,019 bp long, with a large single-copy region of 85,910 bp and a small single-copy region of 19,247 bp separated by two inverted repeats of 26,431 bp. It encodes 130 genes, including 85 protein-coding genes, 37 tRNA genes, and eight ribosomal RNA genes. The phylogenetic analysis indicated that P. conradinae is closely related to the congeners P. maximowiczii, P. takesimensis, P. speciosa, P. serrulata var. spontanea, P. discoidea, and P. matuurai.