Molecular phylogeny in Indian Citrus L. (Rutaceae) inferred through PCR-RFLP and trnL-trnF sequence data of chloroplast DNA

The taxonomy and phylogeny of Indian Citrus is revisited using PCR-RFLP of the trnD-trnT and rbcL-ORF 106 regions as well as sequence data analysis of the trnL-trnF intergenic spacer region of cpDNA. The study was based on 50 accessions of Citrus genotypes, collected from wild, semi-wild and domesticated stocks. Of the 13 restriction enzymes (RE) used for restriction digestion of the polymerase chain reaction (PCR) amplicons, four (Hinf I, Msp I, Alu I, Hae III) generated 47 restriction fragments, of which 24 (51%) were polymorphic. PCR-RFLP data showed a genetic distance ranging from 0 to 0.79 among 50 accessions of Citrus, and a cluster analysis, based on Neighbor-Joining (NJ) method, placed all the accessions in eight major clusters. Analysis of trnL-trnF sequences from 23 representative accessions of Citrus showed a pair-wise sequence divergence rate in the range of 0–0.064. NJ, minimum evolution (ME) and maximum parsimony (MP) analyses of trnL-trnF sequences produced phylogenetic trees, which placed all the 23 accessions in five clusters. PCR-RFLP analysis resulted in a well resolved phylogenetic tree with branches supported by moderate to high bootstrap values, while the trnL-trnF sequence-based trees showed only moderate to low bootstrap support for the internal tree branches, indicating uncertain origin of some Citrus genotypes. This study shows that the trnL-trnF spacer sequence data can detect genetic variation in Indian Citrus genotypes, but the utility of the data in inferring phylogeny at intra and inter-specific levels is limited probably by factors such as hybridization, bud mutations, apomixis and polyploidy. However, PCR-RFLP and trnL-trnF data supported the recognition of C. maxima, C. medica, and C. reticulata as the basal species of edible Citrus.     

Aroma volatiles emission in relation to chilling injury in ‘Kensington Pride’ mango fruit

Summary

Mature green mango fruit (Mangifera indica L. ‘Kensington Pride’) were stored at 0, 5, 10, 15 or 20°C for 1, 3, 7, 14, 21 or 28 d to induce different levels of chilling injury (CI) and to elucidate its relationship with aroma volatile production. The fruit were removed from storage and allowed to ripen at 22 ± 1°C and CI index was assessed on fully ripe fruit. Aroma volatile compounds were estimated from the pulp of fully ripe fruit. CI index significantly increased as the storage temperature was lowered and the storage period was extended, particularly in the fruit stored at 0, 5 or 10°C. CI symptoms did not develop on fruit stored at 15 or 20°C. Fifty-six aroma volatile compounds were identified from mango fruit pulp using headspace solid phase microextraction (SPME) technique with gas chromatography (GC-FID) and GC combined with mass spectrometry (GC-MS). Among the 56 aroma volatile compounds, 25 were quantified using GC, which included monoterpenes (±-pinene, ²-pinene, myrcene, 2-carene, ±-phellandrene, 3-carene, ±-terpinene, limonene, ocimine, ³-terpinene, ±-terpinolene, and a-terpineol), sesquiterpenes (±-copaene, ±-gurjunene, trans-carophyllene, aromadendrane, ±-humulene, alloaromadendrane, ³-gurjunene, and ledene), hydrocarbon (p-cymene), esters (methyl octanoate and ethyl caprylate), aldehyde (decanal), and norisoprenoid (²-ionone). A significant reduction in total aroma volatiles, monoterpenes, sesquiterpenes, hydrocarbon, esters, aldehyde, and norisoprenoid production were observed in fruit stored at 0, 5, 10 or 15°C compared with fruit stored at 20°C. The degree of reduction in aroma volatile compounds depended on the severity of CI, induced with different storage temperatures and storage periods. In conclusion lower temperature storage induced CI in mango fruit and adversely affected the aroma volatiles production.     

Protein markers of Marteilia sydneyi infection in Sydney rock oysters, Saccostrea glomerata

Marteilia sydneyi is the causative agent of QX disease in Sydney rock oyster, Saccostrea glomerata . It is responsible for disease outbreaks among oysters that occur during summer and can result in up to 95% mortality. QX disease has significantly decreased S. glomerata production in some areas of Australia's eastern seaboard over the past 30 years. Marteilia sydneyi sporulates in the digestive gland of oysters leading to complete disorganization of the infected tissues. The current study used proteomics to identify potential molecular markers of sporulating M. sydneyi infection during a field trial undertaken in the Georges River, Sydney, between December 2006 and May 2007. Early stages of M. sydneyi infection were detected by polymerase chain reaction, whilst cytological examination was used to identify sporulating M. sydneyi in the gut. Protein expression in oyster haemolymph was assessed during the M. sydneyi infection period by two dimensional electrophoresis. Proteome maps identified significant differences in the expression of four proteins in oysters with sporulating M. sydneyi infections.     

Nutrients removed by Kappaphycus alvarezii (Rhodophyta, Solieriaceae) in integrated cultivation with fishes in re-circulating water

The potential of the red alga Kappaphycus alvarezii to remove nutrients was tested to treat effluents of Trachinotus carolinus fish cultivation, and the production of carrageenan in this condition was analyzed. Experiments were conducted in four tanks of 8000 L with approximately 1200 fishes of 30 g each integrated with three tanks of 100 L with 700 g of K. alvarezii, as initial biomass per tank. Seawater was re-circulated between tanks with seaweed and with fish. As a control, three tanks with seawater circulating in an open system were utilized. Seawater samples were collected daily for 10 days and concentrations of nitrate, nitrite, ammonium and phosphate were determined in the inflow and outflow water of the tanks. Significant differences between both collecting points were considered as nutrient removal by the seaweed. Growth rates and carrageenan yields were also analyzed in seaweed cultivated in seawater and in effluents. Growth rates of seaweed cultivated in tanks were lower than those obtained in open sea and in laboratory cultivation. Effluents had concentrations of nitrate and nitrite ca. 100 times higher than in the control. Maximum values of nutrient removal on effluents were: nitrate = 18.2%; nitrite = 50.8%; ammonium = 70.5% and phosphate = 26.8%. All plants survived throughout the experimental period, but some developed “ice–ice”, a disease associated with physiological stress. After the experimental period, some plants selected and cultivated in open sea presented higher growth rates in 40 days, indicating nutrient storage. No significant differences between carrageenan yields of K. alvarezii cultivated in seawater and in the effluents were observed. Our results show that K. alvarezii can be utilized as a biofilter for fish cultivation effluents, reducing the eutrophication process and can also be processed for carrageenan production, which provides an additional benefit to the fisheries.     

Lipoperoxidation and cyclooxygenases 1 and 2 inhibitory compounds from Iryanthera juruensis

Plants from Iryanthera genus have been traditionally used as food supplements by South American Indians. The MeOH extract of leaves of Iryanthera juruensis, one of the plants endemic to the Amazon region and consumed in Brazil, and the hexane extract from its seeds inhibited lipid peroxidation (LPO) and cyclooxygenase (COX-1 and -2)) enzymes in in vitro assays. Further analyses of these extracts yielded 5-deoxyflavones (1-5) from the leaf extract and sargachromenol (6), sargaquinoic acid (7), a novel juruenolic acid (8), omega-arylalkanoic acids (9a-c), and the lignan guaiacin (10) from the seed extract. Compounds 3-5 inhibited LPO by 86%, 77%, and 88% at 10 ppm, respectively, and compounds 6 and 9a-c showed inhibition at 76% and 78% at 100 ppm, respectively. However, compounds 7 and 8 were inactive and lignan 10 exhibited LPO inhibitory activity by 99% at 100 ppm compared to commercial antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and vitamin E. The flavones 1-5 also inhibited COX-1 and -2 enzymes by 50-65% at 100 ppm. Compound 6 showed high but nonselective inhibition of COX-1 and COX-2 enzymes, when compared to aspirin and Celebrex, a nonsteroidal anti-inflammatory drug (NSAID). Compounds 7 and 10 inhibited COX-1 by 60% and 65% and COX-2 by 37% and 18%, respectively, whereas compounds 8 and 9a-c showed little or no activity against these enzymes.     

Biochemical and Microbiological Assessment of Crucifix Crab (Charybdis feriatus) Stored at 4°C

Charybdis feriatus is a highly prized crab in the local and international markets of East Asian countries. In local markets, crabs are sold live, iced, or refrigerated at 2°C – 4°C. The present study was carried out to determine the shelf life of whole crab stored at 4°C for 192 h using a set of biochemical, microbiological, and sensorial parameters. Biochemical indicators such as the total volatile base nitrogen (TVB-N) and trimethylamine nitrogen (TMA-N) increased significantly during storage and within an acceptable limit up to 144 h. Regarding the biogenic amines, cadaverine and spermidine increased significantly (p < 0.05) during the storage period. Among them, cadaverine could be proposed as a useful spoilage indicator for this crab species. The mesophilic and psychotropic bacterial count exceeded the limit of acceptance after 144 h of storage. Sensory attributes were gradually diminished with the storage time. Sensory scores were found to correlate well with both mesophiles and psychrotrophs. Based on the results of this study, the meat quality of Charybdis feriatus can be maintained up to 144 h when properly stored at a temperature of 4°C ± 1°C.     

Expression of a truncated Pasteurella multocida toxin antigen in Bordetella bronchiseptica

Mild or subclinical respiratory infections caused by Bordetella bronchiseptica are widespread in pigs despite multiple control efforts. Infection with virulent B. bronchiseptica strains is a common risk factor in the establishment of toxin-producing strains of Pasteurella multocida in the nasal cavity of pigs leading to the disease, atrophic rhinitis (AR). This study was designed to explore the possibility of expressing a protective epitope of P. multocida toxin (PMT) in B. bronchiseptica to create single-component mucosal vaccine to control atrophic rhinitis in pigs. To achieve this, a P. multocida toxin fragment (PMTCE), that was non-toxic and protective against lethal challenge in mice, was cloned into a broad-host-range plasmid, PBBR1MCS2, and introduced into B. bronchiseptica by electroporation. The Pasteurella gene construct was placed under the regulatory control of a promoter region that was separately isolated from B. bronchiseptica and appears to be part of the heat shock protein gene family. B. bronchiseptica harboring the plasmid under antibiotic selection expressed the 80kDa PMTCE as determined by PAGE and Western blot with a PMT-specific monoclonal antibody. When introduced into the respiratory tracts of mice, B. bronchiseptica harboring the plasmid construct was reisolated in declining numbers for 72h post-inoculation. Antibody responses (IgM, IgA and IgG) to B. bronchiseptica were detected in serum and respiratory lavage, but PMTCE-specific antibodies were not detected. While further refinements of PMT expression in B. bronchiseptica are necessary, this study provides a basis for the development of a single-component, live-attenuated vaccine against atrophic rhinitis.     

Carbon dioxide assimilation in urea‐treated wheat leaves

Application of 10 mM urea to the flag leaf of wheat plants enhanced in vivo urease activity several fold. Photosynthetic rate was also increased considerably. There were significant differences in the leaf internal carbon dioxide (CO₂) concentrations between the urea‐treated and untreated leaves. The finding that carbon (¹⁴C) was detected in the ethanol extract of the leaves fed with ¹⁴C‐urea suggests that CO₂ released from urea is re‐fixed by the leaves.