Transfection systems for Babesia bovis: A review of methods for the transient and stable expression of exogenous genes

With the recently sequenced Babesia bovis genome, a large pool of genes with unknown function was identified. The ability to complement and knock-out both unknown and previously identified genes would be a valuable tool to better understand gene function in B. bovis parasites. This review describes recent advances in the development of transient and stable transfection systems for B. bovis. Transient transfection constructs were initially generated using the promoter and the 3′ region of the rap-1 genes of B. bovis controlling expression of luciferase as a reporter. Successful expression of luciferase in B. bovis parasites using this plasmid introduced by classic electroporation of B. bovis infected erythrocytes was followed by the identification and characterization of stronger promoters, such as the ef-1α promoter, using transient transfection techniques. Further refinement of the transient transfection technique included development of the ability to transfect free merozoites using nucleofection, an alternative method to electroporation that results in higher transfection yields and improved viability of transfected parasites. Availability of the transient transfection system was critical for the further development of a stable transfection technique using a plasmid designed to target integration of a gfp-bsd gene into the B. bovis ef-1α locus. Several parasite lines resistant to the anti-babesial drug blasticidin (bsd) and constitutively expressing the gfp-bsd gene were generated after transfection. Integration of the gfp-bsd cassette into the genome was demonstrated by Southern blot and sequence analysis. Taken together these experiments demonstrated the feasibility to introduce, integrate and express exogenous genes in B. bovis. The stable transfection protocol was reproducible and used to transfect at least two distinct B. bovis strains. Further development of these transfection systems will facilitate functional analysis of B. bovis genes and will improve our understanding of the biology of and immunological response to this parasite.     

Quantitative trait loci analysis of flowering-time-related traits in tomato

The regulation of flowering time has been studied mainly in photoperiod-sensitive model plants. Little is known about the environmental and genetic regulation of flowering time in photoperiod-insensitive plants. Here, we studied the genetic control of flowering time in day-neutral tomato using quantitative trait locus (QTL) analysis. We used a BC₁F₆ population developed from Solanum lycopersicum ‘M570018’ and its wild relative Solanum pimpinellifolium (PI124039) to measure days to flowering (DTF) and number of leaves preceding the first inflorescence (LN), as well as their underlying developmental processes, which include the number of leaves initiated (LI), percentage of flower-induced plants (reproductive index, RI), days to macroscopic flower bud appearance (DMB) and flower development duration (FDD: DTF − DMB). A composite interval mapping detected 12 QTLs for the six traits, which included two QTLs for DTF on chromosomes 1 and 6. The DTF QTLs explained 43% of the phenotypic variation of this trait. The presence of S. pimpinellifolium alleles in the detected QTLs increased the rate of leaf initiation, reduced LN and hastened flower induction, floral development and anthesis. Most QTLs for LN, LI, RI, DMB and FDD clustered with the DTF QTLs; dmb1, fdd1, li_14d1, li_19d1 and ri_19d1 clustered with dtf1 on chromosome 1, and ln6 and fdd6 clustered with dtf6 on chromosome 6. These results suggest that the QTLs on chromosomes 1 and 6 may form a functional “gene cluster” that drives tomato flowering in synergy. Alternatively, the two DTF QTLs may act as “master genes” that control flowering time through pleiotropic effects on multiple developmental processes.     

彼得·史密斯:这是我熟悉的领域

虽然他不是出色的职业网球运动员,但是没有人比他更熟悉网球教练这个领域。他将休伊特带上巅峰,同时也是彼得·卡特(PETER CARTER,费德勒前教练,后因车祸去世)的教练。彼得·史密斯(PETER SMITH)无疑是近四十年澳洲网球发展史上的大人物。     

The effects of overtraining on blood volumes in standardbred racehorses

Red blood cell hypervolaemia has been used for diagnosis of overtraining in racehorses, and has been suggested as a mechanism of this cause of loss of racing performance. The effects of overload training (OLT) on the plasma, blood and red cell volumes were investigated in a prospective study in 12 Standardbred horses. Measurements of blood volumes were made after eight and 32 weeks of an exercise training study. Horses were randomly allocated to OLT and control groups (n=6) after 16 weeks of training. Training duration and intensity were increased more rapidly for the OLT group from week 16, until overtraining was diagnosed in week 32.There were no significant effects of OLT on plasma, blood or total red cell volumes between weeks eight and 32. These volumes significantly decreased with time. Maximal haematocrit after exercise was lower (P<0.05) in the OT group in week 32 (0.57+/-0.003% L/L) than in week eight (0.59+/-0.004 L/L). It was concluded that red cell hypervolaemia was not a mechanism for the decrease in capacity for exercise that occurs with overtraining.     

A review of potential image fusion methods for remote sensing-based irrigation management: part II

Satellite-based sensors provide data at either greater spectral and coarser spatial resolutions or lower spectral and finer spatial resolutions due to complementary spectral and spatial characteristics of optical sensor systems. In order to overcome this limitation, image fusion has been suggested to obtain higher spatial and spectral resolution images at the same time. Image fusion has been a valuable technique in digital image analysis and comparison because of the availability of multi-spatial and multispectral images from satellite and airborne sensors. It has been applied to merge coarser spatial resolution of multispectral images with a finer spatial resolution panchromatic image to enhance visual apprehension and to provide images that are more informative. Part I companion paper presented and discussed the image downscaling methods. In this paper (part II), the main objective is to review existing image fusion methods for their capability to downscale coarser spatial resolution images for irrigation management applications. A literature review indicated that image fusion methods have not been actively used in obtaining high-resolution land surface temperature (LST) and evapotranspiration (ET) images for irrigation management. However, there is a great potential for applying image fusion methods to retrieve finer LST and ET images from coarser thermal images by fusing them with finer non-thermal color or panchromatic images for irrigation scheduling and management purposes.     

Development of a PCR test to detect the downy mildew causal agent Plasmopara halstedii in sunflower seeds

Plasmopara halstedii , the causal agent of downy mildew of sunflower, is an obligate parasite but viable sporangia and oospores of the pathogen may be found in a quiescent state in seeds of sunflower and therefore may be transported with sunflower seeds in international commercial exchanges. In order to prevent the spread of this pathogen, especially the introduction of potentially new races, an efficient method to analyse sunflower seed samples is required. In this study, a P. halstedii -specific polymerase chain reaction (PCR) test was developed based on the ribosomal large sub unit (LSU) DNA. The forward (PHAL-F) and reverse (PHAL-R) PCR primers were designed from two polymorphic regions of LSU. After screening 22 isolates of P. halstedii corresponding to different races and countries and 32 other oomycete, deuteromycete and ascomycete isolates, the PHAL-F/R primers amplified only a single PCR band of c. 310 bp from P. halstedii . The PHAL-F/R PCR test could detect as little as 3 pg of P. halstedii genomic DNA per 20  µ L reaction volume and enabled the direct detection of P. halstedii in 35 g sunflower seed samples without the need for any prior biological baiting step. An internal amplification control (IAC) was developed to help discriminate against false negative samples due to the potential presence of inhibitory compounds in DNA extracts. The test was successfully used on samples of naturally contaminated seeds. These new molecular tools should be of great interest for quarantine seed testing purposes.     

A monitoring programme for 1,3-dichloropropene and metabolites in groundwater in five EU countries

BACKGROUND: 1,3 Dichloropropene (1,3-D) is a preplanting soil fumigant for the control of cyst and free-living nematodes and is currently undergoing a resubmission under Annex 1 listing of Directive 91/414/EEC. The characteristics of 1,3-D are such that the risk of it or its soil metabolites leaching through the soil profile cannot be excluded. As such, groundwater monitoring programmes were established in five EU countries representing a wide range of agricultural, climatic and hydrogeological situations, covering a range of groundwater vulnerability scenarios. All monitoring was conducted in areas where there has been historical use of 1,3-D. RESULTS: Over 5000 groundwater samples were analysed for the presence of 1,3-D and its metabolites over a 2 year period. Almost all analyses (for parent and metabolites) yielded concentrations of <0.1 microg L(-1). There were just two detections of >0.1 microg L(-1) (0.12 microg L(-1) and 0.4 microg L(-1)) for the 3-chloroacrylic acid metabolite in shallow groundwater samples of the alluvial gravels of the River Tiétar in the Caceres region of Spain. CONCLUSION: Groundwater monitoring programmes have been conducted in the EU in five countries. These have demonstrated that there is negligible contamination of groundwater with 1,3-D or its metabolites across a range of agroclimatic regions where 1,3-D is known to have been used for a number of years. Local scientific knowledge of geological features, hydrology, agricultural practice and specific local issues was essential to the conduct of the study.     

Accumulation of toxigenic <Emphasis Type="Italic">Fusarium</Emphasis> species and <Emphasis Type="Italic">Stenocarpella maydis</Emphasis> in maize grain grown under different cropping systems

Entomopathogenic nematodes in the genus Steinernema are associated with Xenorhabdus spp. bacteria. When steinernematid colonise an insect host the nematode-bacterium association overcomes the insect immune system and kills the host within 48 h. Xenorhabdus spp. produce secondary metabolites that are antifungal to protect nematode-infected cadavers from fungal colonization. The concentrated, or cell-free metabolites of X. szentirmaii exhibit high toxicity against various fungal plant pathogens and show potential as natural bio-fungicides. In the current study, we determined 1) thermo-stability, 2) dose-response, and 3) shelf-life of antifungal metabolites of X. szentirmaii against Monilinia fructicola (cause of brown rot of peach and other stone fruit) and Glomerella cingulata (cause of antharacnose). Thermo-stability was determined by autoclaving bacterial culture broths (121 °C and 15 psi for 15 min) and measuring fungal growth on in potato dextrose agar (PDA) containing 10% of the supernatants. Autoclaving had no impact on the antifungal activity of the secondary metabolites. Over a test period of 9 months, the activity of both extract types did not decline when stored at 4 or 20 °C. A dose-response study (10, 20, 40, 60, 80 and 100% supernatant-containing metabolite) using both phytopathogens demonstrated that a greater dose of supernatant increased antifungal activity. The antifungal-metabolite containing supernatant of X. szentirmaii has potential as a bio-fungicide. These results demonstrate the metabolite(s) are thermo-stable, they have a long shelf-life and require no stabilizing formulation, even at room temperature.