Development of Molecular Marker to Detect Citrus Melanose Caused by Diaporthe citri from Citrus Melanose-like Symptoms.

It is difficult to distinguish melanose and melanoses-like symptoms with the naked eye because they appear similar. To accurately detect melanose symptoms caused by Diaporthe citri from melanose-like symptoms, we developed PCR-based specific primers Dcitri by aligning the internal transcribed spacer (ITS) region of D. citri with the ITS of Diaporthe cytosporella, Diaporthe foeniculina, Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria citri, and Fusarium oxysporum found on citrus peel. PCR results showed that the specific product was amplified in D. citri but not in other isolates including, C. gloeosporioides, B. cinerea, A. citri, F. oxysporum. In addition, specific products were observed in melanose symptoms caused by D. citri but not in melanose-like symptoms, such as copper-injury, sunscald, damages by yellow tea thrips, and pink citrus rust mite. Using the Dcitri primers developed in this study, it is expected that melanose caused by D. citri could be accurately distinguished from melanose-like symptoms.

Different Phytohormonal Responses on Satsuma Mandarin (Citrus unshiu) Leaves Infected with Host-Compatible or Host-Incompatible Elsinoë fawcettii.

Citrus scab, caused by the fungal pathogen Elsinoë fawcettii, is one of the most important fungal diseases affecting Citrus spp. Citrus scab affects young tissues, including the leaves, twigs, and fruits, and produces severe fruit blemishes that reduce the market value of fresh fruits. To study the molecular responses of satsuma mandarin (C. unshiu) to E. fawcettii, plant hormone-related gene expression was analyzed in response to host-compatible (SM16-1) and host-incompatible (DAR70024) isolates. In the early phase of infection by E. fawcettii, jasmonic acid- and salicylic acid-related gene expression was induced in response to infection with the compatible isolate. However, as symptoms advanced during the late phase of the infection, the jasmonic acid- and salicylic acid-related gene expression was downregulated. The gene expression patterns were compared between compatible and incompatible interactions. As scabs were accompanied by altered tissue growth surrounding the infection site, we conducted gibberellic acid- and abscisic acid-related gene expression analysis and assessed the content of these acids during scab symptom development. Our results showed that gibberellic and abscisic acid-related gene expression and hormonal changes were reduced and induced in response to the infection, respectively. Accordingly, we propose that jasmonic and salicylic acids play a role in the early response to citrus scab, whereas gibberellic and abscisic acids participate in symptom development.

Symptomatology of Citrus mosaic sadwavirus (CiMV) in Some Citrus Cultivars and Effect of CiMV Infection on Citrus Fruit Quality.

Citrus mosaic sadwavirus (CiMV) is a closely related virus with the Satsuma dwarf virus (SDV) along with Navel orange infectious mottling virus (NIMV), Natsudaidai dwarf virus (NDV), and Hyugagatsu virus (HV). The present study found that the typical symptoms of CiMV-infected citrus fruits include the appearance of dark blue speckles or ringspots on fruit rinds and the browning of oil glands in the spots as rind coloring began. As rind coloring progressed, the spots gradually faded, whereas the browning of the oil glands worsened to the point that the tissues surrounding the oil glands became necrotic. In very early satsuma mandarins (Citrus unshiu 'Miyamoto Wase') and 'Setoka' cultivar (C. hybrid 'Setoka') of late-maturity citrus, the symptomatic fruits were eventually dropped. And in early satsuma mandarin (C. unshiu 'Miyakawa Wase'), the peel hardness of the virus-infected fruit (1,618.3 ± 305.5, g-force) was more than twice as hard as that of the healthy fruit (636.5 ± 39.1, g-force). The ratio of flesh weight to total fruit weight was higher for the healthy fruit (77.3 ± 1.7%) than for the infected fruit (70.7 ± 0.6) and peel puffing was more severe in the infected fruit (2.9 ± 0.4 mm) than in the healthy fruit (0.9 ± 0.2 mm). The soluble solids content in infected citrus fruits was less values than the healthy fruit by 0.5-1.5 °Brix. These findings reveal that CiMV infection on citrus trees reduces the fruit quality of citrus.

Draft Genome Sequences of Elsinoë fawcettii and Elsinoë australis Causing Scab Diseases on Citrus.

Elsinoë fawcettii and E. australis (phylum Ascomycota) are phytopathogenic fungi causing scab diseases on citrus plants. We report here the high-quality draft genome sequences and ab initio gene predictions of two E. fawcettii strains and one E. australis strain, which differ in their host range. This genome sequence information will provide valuable resources to underpin genomic attributes for determining host range through comparative genomic analyses of citrus scab fungi.

Biocontrol of Citrus Canker Disease Caused by Xanthomonas citri subsp. citri Using an Endophytic Bacillus thuringiensis.

Citrus canker is a devastating disease of citrus caused by Xanthomonas citri subsp. citri (Xcc). A total of 134 endophytic bacteria were isolated from various gymnospermic and angiospermic plants. They were screened for their antagonistic activities against three wild-type and six streptomycin-resistant Xcc strains. TbL-22 and TbL-26, both later identified as Bacillus thuringiensis, inhibited all the wild and resistant Xcc strains. TbL-22 exerted the highest antagonistic activity against XccW3 and XccM6 with inhibition zones of 20.64 ± 0.69 and 19.91 ± 0.87 mm, respectively. Similarly ethyl acetate extract of TbL-22 showed highest inhibition zones 15.31 ± 2.08 and 19.37 ± 3.17 mm against XccW3 and XccM6, respectively. TbL-22 reduced canker incidence on infected leaves by 64.05% relative to positive controls. Scanning electron microscopy revealed that the cell membranes of Xcc treated with ethyl acetate extract of TbL-22 were ruptured, lysed, and swollen. B. thuringiensis TbL-22 can effectively and sustainably controls streptomycin-resistant citrus canker.

Loop-mediated Isothermal Amplification assay for Detection of Candidatus Liberibacter Asiaticus, a Causal Agent of Citrus Huanglongbing.

Huanglongbing (HLB, Citrus greening disease) is one of the most devastating diseases that threaten citrus production worldwide. Although HLB presents systemically, low titer and uneven distribution of these bacteria within infected plants can make reliable detection difficult. It was known loop-mediated isothermal amplification (LAMP) method has the advantages of being highly specific, rapid, efficient, and laborsaving for detection of plant pathogens. We developed a new LAMP method targeting gene contained tandem repeat for more rapid and sensitive detection of Candidatus Liberibacter asiaticus (CLas), putative causal agent of the citrus huanglongbing. This new LAMP method was 10 folds more sensitive than conventional PCR in detecting the HLB pathogen and similar to that of real-time PCR in visual detection assay by adding SYBR Green I to mixture and 1% agarose gel electrophoresis. Positive reactions were achieved in reaction temperature 57, 60 and 62°C but not 65°C. Although this LAMP method was not more sensitive than real-time PCR, it does not require a thermocycler for amplification or agarose gel electrophoresis for resolution. Thus, we expect that this LAMP method shows strong promise as a reliable, rapid, and cost-effective method of detecting the CLas in citrus and can be applied for rapid diagnosis is needed.

Development of Multiplex PCR for Simultaneous Detection of Citrus Viruses and the Incidence of Citrus Viral Diseases in Late-Maturity Citrus Trees in Jeju Island.

Satsuma dwarf virus (SDV) or Citrus mosaic sadwavirus (CiMV) were not consistently detected in RT-PCR assay with the primer sets based on gene of Japan isolates. SDV and CiMV isolates were distinctively divided into two groups based on phylogenetic analysis of PP2 gene cloned from 22 Korean isolates, and the Korean CiMV and SDV isolates shared 95.5-96.2% and 97.1-97.7% sequence identity with Japanese isolate, respectively. We developed PP2-1 primer set based on the PP2 gene sequence of Korean isolates to simultaneously and effectively detect SDV and CiMV. And CTLV-2013 and CTV-po primer sets were newly designed for detection of Citrus tatter leaf virus (CTLV) and Citrus tristeza virus (CTV), respectively. Using these primer sets, a new multiplex PCR assay was developed as a means to simultaneously detect 4 citrus viruses, CTV, CTLV, SDV, and CiMV. The degree of detection by the multiplex PCR were consistent with those of uniplex RT-PCR for detection of each of the viruses. Therefore, the new multiplex PCR provides an efficient method for detecting 4 citrus viruses, which will help diagnose many citrus plants at the same time. We verified that 35.2% and 72.1% of 775 trees in 155 orchards were infected with SDV or CiMV (SDV/CiMV) and CTV by the multiplex-PCR assay, respectively, and CTLV was not detected in any of the trees tested.

Selection of Small Synthetic Antimicrobial Peptides Inhibiting Xanthomonas citri subsp. citri Causing Citrus Canker.

Citrus canker disease decreases the fruit quality and yield significantly, furthermore, emerging of streptomycin-resistant pathogens threatens the citrus industry seriously because of a lack of proper control agents. Small synthetic antimicrobial peptides (AMPs) could be a promising alternative. Fourteen hexapeptides were selected by using positional scanning of synthetic peptide combinatorial libraries. Each hexapeptide showed different antimicrobial spectrum against Bacillus, Pseudomonas, Xanthomonas, and Candida species. Intriguingly, BHC10 showed bactericidal activity exclusively on Xanthomonas citri subsp. citri (Xcc), while BHC7 was none-active exclusively against two Pseudomonas spp. at concentration of 100 µg/ml suggesting potential selectivity constrained in hexapeptide frame. Three hexapeptides, BHC02, 06 and 11, showed bactericidal activities against various Xcc strains at concentration of 10 µg/ml. When they were co-infiltrated with pathogens into citrus leaves the disease progress was suppressed significantly. Further study would be needed to confirm the actual disease control capacity of the selected hexapeptides.

Flavonoids and a Limonoid from the Fruits of Citrus unshiu and Their Biological Activity.

The fruits of Citrus unshiu are one of the most popular and most enjoyed fruits in Korea. As we continue to seek for bioactive metabolites from Korean natural resources, our study on the chemical constituents of the fruits of C. unshiu resulted in the isolation of a new flavonoid glycoside, limocitrunshin 1, along with seven other flavonoids 2-8 and a limonoid 9. All structures were identified by spectroscopic methods, namely 1D and 2D NMR, including HSQC, HMBC, and (1)H-(1)H COSY experiments, HRMS, and other chemical methods. Compounds 3, 5, and 9 are reported to be isolated from this fruit for the first time. The isolated compounds were applied to activity tests to verify their inhibitory effects on inflammation and nephrotoxicity. Compounds 6 and 9 showed the most potent inhibitory activity on renal cell damage and nitric oxide production, respectively. Thus, the fruits of C. unshiu could serve as a valuable natural source of bioactive components with health benefits for potential application in functional foods.

Physical Changes in Satsuma Mandarin Leaf after Infection of Elsinoë fawcettii Causing Citrus Scab Disease.

Citrus scab disease is one of the destructive diseases that reduce the value of fruit for the fresh market. We analyzed the process of symptom development after infection with scab pathogen Elsinoë fawcettii in the susceptible satsuma mandarin leaves to observe the structural modification against pathogen. The cuticle and epidermal cells along with 3-5 layers of mesophyll tissue were degraded 1-2 days post inoculation. Surrounding peripheral cells of degraded tissues grew rapidly and then enveloped the necrotic area along with the growing conidia. Cross sections through the lesion revealed hyphal colonization in epidermis and mesophyll tissues. In response to the pathogen colonization, host cell walls were lignified, inner cells were rapidly compartmentalized and a semi-circular boundary was formed that separated the infected region from the non-infected region, and finally prevented the intercellular pathogen spread.

Anti-inflammatory activity of a new cyclic peptide, citrusin XI, isolated from the fruits of Citrus unshiu.

ETHNOPHARMACOLOGICAL RELEVANCE: Citrus unshiu (Rutaceae) is an easy-peeling citrus fruit, which has been used as a traditional Korean medicine for improving skin elasticity, relieving fatigue and cough, and preventing bronchitis, flu, and various cancers. However, its active components associated with anti-inflammation and underlying mechanisms remain unknown. In this study, we investigated the active constituents from the fruits of Citrus unshiu and evaluated the anti-inflammatory activity in order to support the traditional usage of Citrus unshiu. MATERIAL AND METHODS: Repeated column chromatography, together with a semi-preparative HPLC purification was used to separate the bioactive constituent from the EtOAc soluble fraction of the EtOH extract of Citrus unshiu fruits. Anti-inflammatory effects of the isolated compounds on lipopolysaccharide (LPS)-induced production of pro-inflammatory mediators were examined using RAW264.7 macrophage cells. RESULTS: A new cyclic peptide, citrusin XI (1), was isolated and identified from the fruits of Citrus unshiu. The structure of compound 1 was elucidated by spectroscopic analysis, including 1D and 2D nuclear magnetic resonance (NMR) ((1)H, (13)C, COSY, HMQC and HMBC experiments), and high resolution (HR)-mass spectrometry, and its absolute configurations were further confirmed by the Marfey׳s method. Compound 1 decreased NO production in LPS-stimulated RAW264.7 cells in a dose-dependent manner with an IC50 value of 70µM. Compound 1 suppressed NO production by decreasing iNOS expression but COX-2 expression was slightly associated with the reduction by compound 1 in LPS-induced RAW264.7 cells. Furthermore, compound 1 inhibited NF-κB activation by blocking IκBα degradation and NF-κB phosphorylation in LPS-stimulated RAW264.7 cells. CONCLUSIONS: These results indicate that a new cyclic peptide, citrusin XI, from Citrus unshiu fruits has anti-inflammatory properties that inhibit the release of pro-inflammatory mediators. Compound 1 decreases NO production by decreasing iNOS expression and NF-κB activation associated with IκBα degradation and NF-κB phosphorylation in LPS-induced RAW264.7 cells. This is the first study to clarify the underlying mechanism of the anti-inflammatory effect exerted by a pure isolated compound from Citrus unshiu in LPS-stimulated RAW264.7 macrophage cells. The phytochemical, citrusin XI of Citrus unshiu may serve as lead compound in the design of new agents for preventing and treating inflammatory diseases.

Characterization and in vitro inhibition studies of Bacillus anthracis FtsZ: a potential antibacterial target.

FtsZ is an essential bacterial cell division protein that is an attractive target for the development of antibacterial agents. FtsZ is a homologue of eukaryotic tubulin, has GTPase activity, and forms a ring-type structure to initiate cell division. In this study, the FtsZ of Bacillus anthracis was cloned into a bacterial expression vector and overexpressed into Escherichia coli BL21 (DE3) cells. The overexpressed B. anthracis FtsZ was soluble and purified to homogeneity using Ni-His-tag affinity chromatography. Like other known FtsZs, the recombinant B. anthracis FtsZ also showed GTP-dependent polymerization, which was analyzed using both spectrophotometric and Transmission Electronic Microscopic (TEM) analysis. Using the purified FtsZ, we screened a naturally extracted chemical library to identify potent and novel inhibitors. The screening yielded three chemicals, SA-011, SA-059, and SA-069, that inhibited the in vitro polymerization activity of FtsZ in the micromolar range (IC50 of 55-168 µM). The inhibition potency was significantly comparable with that of berberine, a known potential inhibitor of FtsZ. Understanding the biochemical basis of the effect of these inhibitors on B. anthracis growth would provide a promising path for the development of new antianthracis drugs.

Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit.

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3µM, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1µM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7Å distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.

Comparative ultrastructure of nonwounded Mexican lime and Yuzu leaves infected with the citrus canker bacterium Xanthomonas citri pv. citri.

Ultrastructural aspects of citrus canker development were investigated in nonwounded leaves of citrus species by transmission electron microscopy (TEM). A susceptible species Mexican lime and a resistant species Yuzu were spray-inoculated with a virulent strain of Xanthomonas citri pv. citri. Initial symptoms occurred on Mexican lime approximately 9 days after inoculation, whereas they appeared on Yuzu mostly 11 days after inoculation. In Mexican lime leaves, the bacterial invasion was usually accompanied by host cell wall dissolution and cellular disruption. Fibrillar materials from degenerated cell walls were usually found in intercellular spaces. Damaged host cells with necrotic cytoplasm showed the localized separation of plasma membrane from the cell wall. Bacterial multiplication and electron-transparent capsule-like structures around bacteria were commonly observed. Meanwhile, cell wall protuberances were prominent outside host cell walls in response to bacterial invasion in Yuzu leaves. Occlusion of intercellular spaces was also formed by the fusion of two or more individual cell wall protuberances originated from two adjacent host cells. Papillae-like materials accumulated locally within host cells in close proximity to bacteria. Some bacteria were found to be undergoing degeneration in xylem vessels. Also, the shrunken, inactive bacteria were surrounded by electron-translucent fibrillar materials in intercellular spaces, implying bacterial immobilization. These cellular responses are thought to be the consequences of defense responses of Yuzu leaves to invading bacteria. In both citrus species, X. citri pv. citri contained polyphosphate bodies showing electron-dense and elliptical structures in cytoplasm.

Nonhost-associated proliferation of intrahyphal hyphae of citrus scab fungus Elsinoe fawcettii: refining the perception of cell-within-a-cell organization.

Ultrastructural aspects of intrahyphal hyphae formation were investigated in Elsinoe fawcettii by transmission electron microscopy. Desiccated and hydrated cultures of E. fawcettii hyphae in liquid and solid media were prepared to determine the effects of water/nutrient availability and media fluidity on the formation of intrahyphal hyphae of the fungus. In all the culture conditions, intrahyphal hyphae were observed in enclosing hyphae. Electron-transparent hyphal cell walls clearly delimited intrahyphal hyphae from the cytoplasm of enclosing hyphae. Intrahyphal hyphae occupied most of the lumen of the enclosing hyphae, and showed intact hyphal cytoplasm with distinct organelles and inclusions. Intrahyphal hyphae were found to grow out of the degenerated hyphae that were almost devoid of cellular contents (simple intrahyphal hyphae). Some intrahyphal hyphae appeared to push aside a septum and passed into the adjacent hyphal cell. Besides a single intrahyphal hypha, instances were noted where enclosing hyphae contained several individual intrahyphal hyphae (multiple intrahyphal hyphae). Other enclosing hyphae contained intrahyphal hyphae, which also had intrahyphal hyphae (compound intrahyphal hyphae). The cell wall of intrahyphal hyphae showed the continuity with the cell wall of the enclosing hyphae. Concentric bodies typical of ascomycetes occurring in dry habitats were not found in all the types of hyphae. These results suggest that intrahyphal hyphae formation of E. fawcettii does not require plant defense responses. The fungus is thought to form intrahyphal hyphae during the saprophytic phase in ex planta ecological niches as well as the parasitic phase in host parts.

Sensitive and specific detection of Xanthomonas axonopodis pv. citri by PCR using pathovar specific primers based on hrpW gene sequences.

A sensitive and specific assay was developed to detect citrus bacterial canker caused by Xanthomonas axonopodis pv. citri, in leaves and fruits of citrus. Primers XACF and XACR from hrpW homologous to pectate lyase, modifying the structure of pectin in plants, were used to amplify a 561 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally or artificially infected leaves of citrus. The PCR product was only produced from X. axonopodis pv. citri among 26 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference microbes.