A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts

Background  

Transient assays using protoplasts are ideal for processing large quantities of genetic data coming out of hi-throughput assays. Previously, protoplasts have routinely been prepared from dicot tissue or cell suspension cultures and yet a good system for rice protoplast isolation and manipulation is lacking.     

Characterization of Small-size Egg-bearing Thai ss-strain Rotifers Brachionus rotundiformis and Their First Offspring

This study selected small (100–120 μm) egg-bearing females from a local Thai ss population and characterized their first offspring's life history in an attempt to produce small-size rotifers. Mean lorica length of selected small adult females was 120 ± 6 μm at the time of collection, and this mean length did not increase over their lifetime. While their first offspring life span was only 5.11 ± 0.35 d, the reference population longevity was higher, 9.65 ± 0.4 d. The length of reproductive period of offspring from the small-size egg-bearing female was 2.58 ± 0.38 d, shorter than that of reference population 6.83 ± 0.58 d. Although the rate of egg production was not different between small egg-bearing female and reference population, the total number of eggs produced by the small egg-bearing female's first offspring was lower (4.78 ± 0.60) compared to the reference population (11.83 ± 1.26). The small egg-bearing female's first offspring had a higher egg to lorica length ratio (76.1 ± 1.64%) than the reference population (56.6 ± 1.3%), indicating a relatively high investment in reproduction. The small egg-bearing female's first offspring reached only 125 ± 6 μm in 36 h, and further culture of their offspring over 35 d resulted in a mean offspring size, 163 ± 11 μm, similar to the reference population (159 ± 16 μm). This shows that population mean size reduction is not likely to result from selecting small egg-bearing females within a single generation. The unique reproductive characteristics of small egg-bearing female place it in its own category.     

Characterization of the Microbial Community Involved in the Suppression of Pythium aphanidermatum in Cucumber Grown on Rockwool

ABSTRACT The root pathogen Pythium aphanidermatum induced lower levels of disease in cucumber (Cucumis sativus) plants on unsterilized, re-used rockwool slabs than on heat-sterilized, re-used rockwool. Several recolonization treatments of the sterilized rockwool enhanced the suppressiveness of the rockwool. Microbial community structures in the different rockwool treatments were investigated by plate counts on selective media. Disease suppressiveness in the different rockwool treatments showed the highest correlation with the culturable number of filamentous actinomycetes in both experiments (r = 0.79 and 0.94), whereas the numbers of Trichoderma spp. correlated with suppression only in the first experiment (0.86). The numbers of total culturable bacteria, fluorescent pseudomonads, Bacillus spores, and fungi all showed lower correlations with disease suppressiveness. The filamentous actinomycetes enumerated with the plate counts were mainly Streptomyces spp., of which 10% were antagonistic toward P. aphanidermatum in dual culture. The composition of the bacterial and actinomycete populations was studied with polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). Multivariate analyses of these patterns with canonical correspondence analysis showed significant correlations between the microbial composition and the disease suppressiveness. However, none of the bands in PCR-DGGE patterns occurred exclusively in the treatments that had enhanced disease suppressiveness. Bands extracted from the actinomycete-specific DGGE gels showed closest similarity with members of several actinomycete genera, i.e., Streptomyces, Mycobacterium, Microbacterium, Rhodococcus, Curtobacterium, and Tsukamurella. The possible mechanism of disease suppressiveness in used rockwool slabs, based on the results obtained with culture-dependent and culture-independent detection methods, is discussed.     

Perfrin,a novel bacteriocin associated with netB positive Clostridium perfringens strains from broilers with necrotic enteritis

Necrotic enteritis in broiler chickens is associated with netB positive Clostridium perfringens type A strains. It is known that C. perfringens strains isolated from outbreaks of necrotic enteritis are more capable of secreting factors inhibiting growth of other C. perfringens strains than strains isolated from the gut of healthy chickens. This characteristic could lead to extensive and selective presence of a strain that contains the genetic make-up enabling to secrete toxins that cause gut lesions. This report describes the discovery, purification, characterization and recombinant expression of a novel bacteriocin, referred to as perfrin, produced by a necrotic enteritis-associated netB-positive C. perfringens strain. Perfrin is a 11.5 kDa C-terminal fragment of a 22.9 kDa protein and showed no sequence homology to any currently known bacteriocin. The 11.5 kDa fragment can be cloned into Escherichia coli, and expression yielded an active peptide. PCR detection of the gene showed its presence in 10 netB-positive C. perfringens strains of broiler origin, and not in other C. perfringens strains tested (isolated from broilers, cattle, sheep, pigs, and humans). Perfrin and NetB are not located on the same genetic element since NetB is plasmid-encoded and perfrin is not. The bacteriocin has bactericidal activity over a wide pH-range but is thermolabile and sensitive to proteolytic digestion (trypsin, proteinase K). C. perfringens bacteriocins, such as perfrin, can be considered as an additional factor involved in the pathogenesis of necrotic enteritis in broilers.