Molecular Characterization of a Distinct Begomovirus Infecting Euphorbia pulcherrima in China

Virus isolate G35 was obtained from Euphorbia pulcherrima showing leaf curl and vein thickening symptoms in Tianyang, Guangxi Province, China. The virus was transmitted by whiteflies to Nicotiana tabacum, Lycopersicon esculentum, Datura stramonium and E. pulcherrima. DNA‐A contains 2746 nucleotides, with two open reading frames (ORFs) in the virion‐sense DNA and four ORFs in the complementary‐sense DNA. When compared with the DNA‐A sequence of other begomoviruses, the total DNA‐A of isolate G35 was most closely related to that of Ageratum enation virus (79.9% sequence identity). However, the deduced coat protein of G35 is most like that of Pepper leaf curl virus from Bangladesh (94.9% amino acid sequence identity), and the AC1 of G35 is most like that of Cotton leaf curl Multan virus‐Okra (87.2% amino acid sequence identity). The molecular data showed that G35 is a distinct Begomovirus species, for which the name Euphorbia leaf curl virus (ELCV) is proposed.     

One‐step Multiplex RT‐PCR for Simultaneous Detection of Four Viruses in Tobacco

A one‐step multiplex RT‐PCR method has been developed for the simultaneous detection of four viruses frequently occurring in tobacco (Cucumber mosaic virus, Tobacco mosaic virus, Tobacco etch virus and Potato virus Y). Four sets of specific primers were designed to work with the same reaction reagents and cycling conditions, resulting in four distinguishable amplicons representative of the four viruses independently. This one‐step multiplex RT‐PCR is consistently specific using different combinations of virus RNA as templates, and no non‐specific band was observed. It has high sensitivity compared to single RT‐PCR. Moreover, field samples in China can be tested by this method for virus detection. Our results show that one‐step multiplex RT‐PCR is a high‐throughput, specific, sensitive method for tobacco virus detection.     

Terpenes from Euphorbia antiquorum and Their in Vitro Anti‐HIV Activity

Three new compounds ( 1 – 3 ), including two euphane type triterpenes, 24,24‐dimethoxy‐25,26,27‐trinoreuphan‐3β‐ol ( 1 ) and (24S)‐24‐hydroperoxyeupha‐8,25‐dien‐3β‐ol ( 2 ), and an ent‐atisine diterpene, ent‐atisane‐3α,16α,17‐triol ( 3 ), were isolated from an acetone extract of the stems of Euphorbia antiquorum, together with eight known diterpenes ( 4 – 11 ). The structures of compounds ( 1 – 11 ) were elucidated using NMR and MS spectroscopic methods. Compound 7 showed moderate activity against HIV‐1 replication in vitro (EC₅₀ = 1.38 μm ).     

Occurrence of Six Begomoviruses Infecting Tomato Fields in Venezuela and Genetic Characterization of Potato Yellow Mosaic Virus Isolates

Begomoviruses are one of the major pathogens in tomato crops worldwide. In Venezuela, six begomovirus species have been described infecting tomato: Potato yellow mosaic virus (PYMV), Euphorbia mosaic Venezuela virus (EuMVV), Merremia mosaic virus (MeMV), Tomato chlorotic leaf distortion virus (ToCLDV), Tomato yellow margin leaf curl virus (TYMLCV) and Tomato yellow leaf curl virus (TYLCV). In this study, the occurrence of these viruses was analysed by PCR in 338 tomato plants exhibiting virus‐like symptoms. Sixty‐three per cent of the plants were positive at least to one of the begomoviruses tested. PYMV and TYLCV were the most frequent viruses showing 39.6 and 23.7% occurrence, respectively. Phylogenetic analyses revealed two groups of PYMV isolates from several Caribbean Basin countries. The first group clustered isolates from several countries, including Venezuela, and the second group clustered only Colombian isolates. Due to the high prevalence of PYMV and TYLCV in Venezuela, it is suggested that the surveillance and control strategies currently applied in the country should be focused on these two begomoviruses.     

Study of mRNA Expression by Real Time PCR of Cpkk1, Cpkk2 and Cpkk3, three MEKs of Cryphonectria parasitica,in Virus‐free and Virus‐infected Isogenic Isolates

Cpkk1 and Cpkk2 are two previously characterized Mitogen‐activated protein kinase kinases (MEK) from Cryphonectria parasitica. For the characterization of the third MEK, primers designed to a conserved region of the known fungal MEK sequences were used in a PCR reaction to amplify genomic DNA from C. parasitica. The sequence of the resulting amplicon was compared to known sequences in the database using a Blast search. Results of the sequence comparison indicated that the initial fragment obtained encoded for a new MEK from C. parasitica, that had highest homology to Pbs2 from Saccharomyces cerevisiae. By inverse PCR we obtained a genomic fragment spanning the entire coding sequence of this MEK, which was named Cpkk3. The cDNA of Cpkk3 was obtained by compiling the sequences of RT‐PCR products resulting from the amplification of purified mRNA. TaqMan^® Probes were designed to analyse the expression of Cpkk1, Cpkk2 and Cpkk3 mRNA through RT‐Real Time PCR. This protocol allowed the expression of Cpkk3 to be successfully compared to the expression of Cpkk1 and Cpkk2, two previously cloned C. parasitica MEKs. No variation in expression was associated with the presence of a virus after 2 days of growth in standard conditions whereas an increase in the expression level of all the three MEKs was shown after 4 days of growth.     

Real‐time PCR detection of Enterococcus faecalis associated with amyloid arthropathy

Aims: To develop a RT‐PCR method for detection of the multilocus sequence type 82 of Enterococcus faecalis associated with amyloid arthropathy (AA) in layers. Methods and Results: Bacteria were selected from lesions including AA in layers. The primers were designed based on the phosphate ATP binding cassette transporter (pstS) and xanthine phosphoribosyltransferase (xpt) genes and first tested against three isolates with known base pairs at the specific sites. Subsequently, 12 isolates were selected from our collection by one researcher, and RT‐PCR was performed blinded. The sequence type (ST) was then confirmed by multilocus sequence analysis. Two single‐nucleotide polymorphisms in the pstS and xpt genes allowed an unambiguous identification of ST82. As an alternative to DNA extraction, a boiling method for release of DNA from cells was used. Conclusions: The real‐time PCR targeting ST82 enables rapid screening of Ent. faecalis cultured from suspect cases with results available after a few hours, much faster than multilocus sequence typing and pulse field gel electrophoresis. Significance and Impact of the Study: The new method allows a rapid screening of isolates with results available after only few hours. This RT‐PCR method could be a useful tool for molecular epidemiological studies on the spread of arthropathic and amyloidogenic Ent. faecalis within and between birds more efficiently.     

Norlathyrane Diterpenes from the Root of Euphorbia kansuensis

Phytochemical investigation of the EtOH extract obtained from the root of the Euphorbia kansuensis Proch . grown in China resulted in the isolation of two novel norlathyrane diterpenes, named ekanpenoids A and B ( 1 and 2 , resp.). Their structures were established by extensive 1D‐ and 2D‐NMR spectroscopy, as well as other spectra. The isolated diterpenes exhibited potent cytotoxic activities against the HeLa and Hep‐G2 cell lines with the IC₅₀ values ranging from 3.6 to 9.7 μg/ml.     

Daphnane‐Type Diterpenoid Glucosides and Further Constituents of Euphorbia pilosa

Phytochemical investigation of whole plants of Euphorbia pilosa led to the isolation and identification of two new daphnane‐diterpenoid glucosides, euphopilosides A and B ( 1 and 2 , resp.), and a new ent‐abietane, euphopilolide ( 3 ), together with eight known compounds. Compounds 1 and 2 are the first daphnane‐type diterpenoid glycosides. Their structures were elucidated by a combination of 1D‐ and 2D‐NMR, and MS analyses, and acid hydrolysis. Compounds 1 – 9 were evaluated for their in vitro cytotoxicities against five human tumor cell lines, HL‐60, SMMC‐7721, A‐549, MCF‐7, and SW‐480. Compound 7 showed moderate inhibitory activity against all five cell lines.     

Interveinal Yellowing Caused by Tomato infectious chlorosis virus in Lettuce and Escarole in Southern Italy

In 2005 and 2006, a severe disease of lettuce and escarole, characterized by interveinal yellowing of the leaves, was observed in the Calabria region (Southern Italy). Samples collected were positive in RT‐PCR assay when specific primers for Crinivirus were used. The identity of the virus was determined by using Tomato infectious chlorosis virus (TICV)‐specific primers and probe. An amplicon of the same size (501 bp) as that from TICV‐infected tomato controls, corresponding to the partial Hsp70 viral gene, was obtained from 36 of 40 lettuce samples and from 19 of 24 escarole samples tested in 2005. The sequences obtained had 99–100% nucleotide sequence identity with other TICV sequences. In an extensive survey carried out in 2006 with TICV‐specific probe, 92% of symptomatic lettuce and 89% of escarole samples were infected with TICV. Adults of Trialeurodes vaporariorum, harvested from symptomatic lettuce and escarole plants, transmitted the virus to healthy plants of the same species. This is the first evidence of a disease caused by TICV in lettuce in Italy and the first record of TICV infection in escarole.     

Extraction and characterization of a natural rubber from Euphorbia characias latex

A natural rubber was identified and characterized for the first time in the latex of the perennial Mediterranean shrub Euphorbia characias. Four different methods, i.e., acetone, acetic acid, trichloroacetic acid, and Triton® X‐100, followed by successive treatments with cyclohexane/ethanol, were employed to extract the natural rubber. The rubber content was shown to be 14% (w/v) of the E. characias latex, a low content compared with that of Hevea brasiliensis (30–35%) but a similar content to other rubber producing plants. E. characias rubber showed a molecular weight of 93,000 with a M_w/M_n of 2.9. ¹H NMR, ¹³C NMR, and FTIR analysis revealed the characteristic of the cis‐1,4‐polyisoprene typical of natural rubber. These results provided novel insight into latex components and will ultimately benefit the broader understanding of E. characias latex composition. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 589–594, 2012.     

Synthesis of New Lathyrane Diterpenoid Derivatives from Euphorbia lathyris and Evaluation of Their Anti‐Inflammatory Activities

Euphorbia factor L₃, a lathyrane diterpenoid extracted from Euphorbia lathyris, was found to display good anti‐inflammatory activity with very low cytotoxicity. To find more potent anti‐inflammatory drugs, two series of Euphorbia factor L₃ derivatives with fatty and aromatic acids were designed and synthesized. Among them, lathyrane derivative 5n exhibited most potent inhibition on LPS‐induced NO production in RAW264.7 cells with no obvious cytotoxicity. To determine the key characteristics of Euphorbia factor L₃ derivatives that contribute to anti‐inflammatory activity, we conducted a structure‐activity relationship study of these compounds.