Monitoring glyphosate residues in transgenic glyphosate-resistant soybean

The availability of Roundup Ready (RR) varieties of soybean has increased the use of glyphosate for weed control in Argentina. Glyphosate [(N-phosphonomethyl)glycine] is employed for the eradication of previous crop vegetation and for weed control during the soybean growing cycle. Its action is effective, and low environmental impact has been reported so far. No residues have been observed in soil or water, either of glyphosate or its metabolite, AMPA (aminomethylphosphonic acid). The objective of this work was to monitor glyphosate and AMPA residues in soybean plants and grains in field crops in Santa Fe Province, Argentina. Five sites were monitored in 1997, 1998 and 1999. Individual soybean plants were sampled from emergence to harvest, dried and ground. Analysis consisted in residue extraction with organic solvents and buffers, agitation, centrifugation, clean-up and HPLC with UV detection. In soybean leaves and stems, glyphosate residues ranged from 1.9 to 4.4 mg kg(-1) and from 0.1 to 1.8 mg kg(-1) in grains. Higher concentrations were detected when glyphosate was sprayed several times during the crop cycle, and when treatments approached the flowering stage. AMPA residues were also detected in leaves and in grains, indicating metabolism of the herbicide.     

Alginate as a new biomaterial for the growth of porcine retinal pigment epithelium

Objective Determine the effect of a 3‐dimensional alginate matrix on the growth and differentiation of cells isolated from porcine retinal pigment epithelium (RPE). Procedures Porcine RPE cells were harvested from enucleated eyecups, isolated by differential gravity sedimentation and cultured in either alginate alone (Group 1) or on plastic tissue culture plates followed by alginate (Group 2). Group 1 cells were cultured in alginate to evaluate the efficacy of the matrix as a culture medium. Group 2 cells were initially cultured on plastic to induce dedifferentiation. The cells were then harvested, suspended in alginate beads, and incubated for a second culture period to determine if the induced dedifferentiation was reversible. Results The number of Group 1 cells was significantly greater (P ≤ 0.01) at the end of the culture period. The amount of pigment and cell morphology of Group 1 cells at the end of the culture period was similar to that seen at initial cell isolation. The initial culture of Group 2 cells on plastic showed characteristic features of dedifferentiation marked by the loss of pigment and alterations in microscopic appearance. Secondary culture of dedifferentiated Group 2 cells in alginate beads resulted in a return to pigmentation and characteristic morphology for a majority of the cultured cells. Conclusions Porcine RPE cells can be propagated in alginate culture with a significant increase in cell numbers while maintaining normal morphology. Under the conditions described in the present study, the dedifferentiation of porcine RPE induced by standard in vitro culture methods is reversible.     

Multiplex Polymerase Chain Reaction Identification of Single Individuals of the Longidorid Nematodes Xiphinema index, X. diversicaudatum, X. vuittenezi, and X. italiae Using Specific Primers from Ribosomal Genes

ABSTRACT The species X. index, X. diversicaudatum, X. vuittenezi, and X. italiae are established (E) or putative (P) vectors of Grapevine fanleaf virus (GFLV) (E), Arabis mosaic virus (E), Grapevine chrome mosaic virus (P), and GFLV (P) nepoviruses of grapevine, respectively. All four species are very closely related taxonomically and their low field densities make them difficult to identify from morphological and morphometrical diagnostic characters when only single or few individuals are detected. To improve diagnostic accuracy, a simple method was developed. The internal transcribed spacer 1 (ITS1) region spanning the 18S and 5.8S ribosomal genes was sequenced in one population of each species using two conserved primers from these genes. The ITS1 fragments were 1,132 bp (X. vuittenezi), 1,153 bp (X. index), 1,175 bp (X. diversicaudatum), and 1,190 bp (X. italiae), i.e., a difference of over 5% between the extremes. The sequence variability made it possible to design species-specific internal sense primers that amplified, in combination with the same antisense ITS1 primer, a single signature fragment (340 bp for X. index, 414 bp for X. italiae, 591 bp for X. vuittenezi, and 813 bp for X. diversicaudatum). Tests with DNA from a single adult or juvenile nematode confirmed the specificity of the primers from diverse isolates or populations. The primers were successfully used in a multiplex test for the reliable detection of two to four mixed species, each represented by a single individual. This multiplex-based diagnostic tool will be particularly useful for successful nematode management practices in vineyards.     

Diagnosis of three <Emphasis Type="Italic">Tospovirus</Emphasis> species by rapid immunofilter paper assay

The rapid immunofilter paper assay (RIPA) was developed to detect Tomato spotted wilt virus (TSWV), Groundnut ringspot virus (GRSV), and Tomato chlorotic spot virus (TCSV) using antisera against recombinant nucleocapsid (N) proteins of each tospovirus. The two-step RIPA was sensitive enough to detect each pecies specifically in only 30min. This technique is proposed as an excellent tool for routine Tospovirus diagnosis and field epidemiological studies.     

United States Department of Agriculture-Agricultural Research Service research on alternatives to methyl bromide: pre-plant and post-harvest

Methyl bromide is a widely used fumigant for both pre-plant and post-harvest pest and pathogen control. The Montreal Protocol and the US Clean Air Act mandate a phase-out of the import and manufacture of methyl bromide, beginning in 2001 and culminating with a complete ban, except for quarantine and certain pre-shipment uses and exempted critical uses, in January 2005. In 1995, ARS built on its existing programs in soil-borne plant pathology and post-harvest entomology and plant pathology to initiate a national research program to develop alternatives to methyl bromide. The focus has been on strawberry, pepper, tomato, perennial and nursery cropping systems for pre-plant methyl bromide use and fresh and durable commodities for post-harvest use. Recently the program has been expanded to include research on alternatives for the ornamental and cut flower cropping systems. An overview of the national research program is presented. Results from four specific research trials are presented, ranging from organic to conventional systems. Good progress on short-term alternatives is being made. These will be used as the foundation of integrated management systems which begin with pre-plant management decisions and continue through post-harvest processing.     

An age-induced switch to a hyper-recombinational state

There is a strong correlation between age and cancer, but the mechanism by which this phenomenon occurs is unclear. We chose Saccharomyces cerevisiae to examine one of the hallmarks of cancer--genomic instability--as a function of cellular age. As diploid yeast mother cells aged, an approximately 100-fold increase in loss of heterozygosity (LOH) occurred. Extending life-span altered neither the onset nor the frequency of age-induced LOH; the switch to hyper-LOH appears to be on its own clock. In young cells, LOH occurs by reciprocal recombination, whereas LOH in old cells was nonreciprocal, occurring predominantly in the old mother's progeny. Thus, nuclear genomes may be inherently unstable with age.