Detecting<Emphasis Type="Italic">Orobanche</Emphasis> species by using cpDNA diagnostic markers

Some species of the genusOrobanche are among the most devastating parasitic weeds, causing extensive damage in agricultural fields. Considering the difficult control due to seed longevity in the soil, small seed size, high fecundity and a subterranean phase that allows them to parasitize the host before they emerge and become evident, the development of diagnostic markers is highly recommended. In our study we identified potential molecular diagnostic markers from the plastid genome in order to distinguish among the most importantOrobanche species attacking crops in Andalusia, the southern region of the Iberian Peninsula. The study has consideredO. crenata, O ramosa andO. cumana causing serious losses in legumes, solanaceous crops and sunflower fields, respectively, andO. minor that, although abundant in Andalusia, has to our knowledge not yet been found parasitizing agricultural hosts. We amplified a non-coding region from the plastid genome, studied sequence differences among the amplified fragments and digested those of the same length with selected restriction enzymes. Here, we propose a molecular protocol to distinguish the main parasitic plants in crop fields of southern Spain. Different applications such as identification ofOrobanche seeds in soil or crop seed lots are discussed in order to offer right crop recommendations or to prevent new infestation of parasite-free fields. Recommendations for further development of these diagnostic markers are also considered. http://www.phytoparasitica.org posting Jan. 15, 2007.     

Factors affecting seed germination and emergence of Gomphrena perennis

Controlled growth chamber experiments were conducted to determine factors affecting seed germination and emergence of the troublesome weed Gomphrena perennis. The objective of this research was to examine the effects of temperature, light, moist chilling, osmotic potential, dry storage and depth of seed burial on G. perennis germination and emergence. The optimum temperature for germination was around 15–20°C. Seeds showed germination rates above 90% under 20/10 and 25/15°C temperature regimes. The minimum exposure to light needed to stimulate germination was 1 min. However, the light requirement was reduced after a long storage period. Furthermore, germination was high (>90%) in all moist‐chilling treatments tested. Germination was highly sensitive to increasing osmotic stress. The highest germination percentage (94%) was achieved at 0 MPa, and decreasing osmotic potential from 0 to ?0.3 MPa reduced germination to 11%. The highest seedling emergence occurred for seeds placed from 0 to 1 cm deep, and no seedlings emerged from a 5‐cm burial depth. Gomphrena perennis has a suitable environment in a no‐till soybean field, where seeds remaining on the surface have the required temperature, light and depth needed for germination.     

Toward a multiplexed serotyping immunoassay for foot-and-mouth disease virus.

Initial results demonstrating the feasibility of a multiplexed liquid array immunoassay for foot-and-mouth disease viral antigen detection and simultaneous serotype differentiation are presented. Serotype-specific antibodies from rabbit and guinea pig hyperimmunesera were isolated and prepared for use in a multiplexed, bead-based assay. The performance of all of the available antibodies as both capture and detector reagents was evaluated in the multiplexed system to establish a combination exhibiting the highest homotypic responses and lowest heterotypic reactions. The multiplexed assay was evaluated against inactivated cell culture supernatant samples of the same subtype as the virus used to raise the capture and detector antibodies. Distinct serotype differentiation was observed, except in the case of serotype SAT1. Subsequently, cell culture supernatant samples from a larger pool of viral subtypes were analyzed. Distinct serotype differentiation was obtained when analyzing cell culture supernatant samples from viral serotypes C, Asia, and SAT3, irrespective of the subtype. However, limitations of the current antibody pairs were realized in some inconclusive results obtained when analyzing samples from a broader range of O, A, and SAT2 subtypes. The results obtained in this initial study will be used to further optimize the assay using polyvalent or monoclonal antibodies and move toward the analysis of clinical samples.     

In vitro plantlet formation from embryonic explants of eastern white cedar (Thuja occidentalis L.)

A protocol is described for the in vitro production of plantlets from embryonic explants of eastern white cedar (Thuja occidentalis L.). Bud induction was optimal when embryonic explants were cultured for 20-25 days on half-strength Quoirin and Le Poivre mineral salts containing equimolar concentrations (10(-6) M) of N(6)-benzyladenine and 2-isopentyl adenine. Bud development was achieved in phytohormone-free medium in the presence of activated charcoal. Maximal shoot elongation occurred on half-strength Quoirin and Le Poivre salts, whereas shoot multiplication was optimal on half-strength Bornman's MCM salts in the presence of cytokinin. Hardened shoots, dipped in commercial rooting powder containing indole-3-butyric acid, rooted optimally in mist under non-sterile greenhouse conditions. Both rooting and subsequent plantlet growth was best when Redi-Earth((R)) was used as a substrate. Over 250 plantlets per embryo can be produced annually by this technique.     

Surface molecules identify groups of growing axons

Studies on vertebrate and invertebrate species have established that, during development, axons have the ability to choose particular paths over others. The chemical basis of this pathfinding is not clear but biochemical differences between neurons have long been postulated to account for the specificity of neuronal connections. Such subtle molecular differences between different cells in a single tissue are difficult to study with standard biochemical techniques but hybridoma technology has offered a potential solution to this type of problem. This technique has made possible the production of monoclonal antibodies for identifying and characterizing a family of glycoproteins which are expressed on the surface of specific axon bundles during the development of the leech nervous system. The results show that groups of growing axons do indeed carry chemically distinct surface molecules.     

Effects of two different neem products on different stages of Nezara viridula (L.) (Heteroptera, Pentatomidae)

In this study, the effects of two different commercial neem insecticides (NeemAzal T/S and Neem Oil) were determined on different stages of Nezara viridula (L.) (Heteroptera: Pentatomidae) under laboratory conditions. Neem Azal and Neem Oil were applied at concentrations of 0.5 % and 2 %, respectively. Mortality was recorded after 3, 7 and 14 days for nymphs and adults; 7 and 14 days for old (4-day-old) eggs; and 14 days for newly laid (one-day-old) eggs. Both products have no significant effect on adults and newly laid eggs. However Neem Oil was found to be more effective than NeemAzal T/S on nymphs and on old laid eggs after 7 and 14 days. It can be concluded that both neem products have potential for insecticidal efficacy (approximately 60 %) against nymphs of N. viridula. at concentrations recommended by manufacturers for registered pests.     

Isolation of Escherichia coli O157:H7 from the gall bladder of inoculated and naturally-infected cattle

To determine if Escherichia coli O157:H7 is capable of residing in the gall bladder of cattle, inoculation studies were conducted with O157:H7 strain 86-24 in weaned Holstein calves. Strain 86-24 was isolated from the gall bladders of five calves 36 days after inoculation. Two other calves contained the inoculation strain in the distal colon but the organism was absent in their gall bladders. A second trial in which the calves were euthanized 15 days after inoculation found strain 86-24 in six of seven inoculated calves but only in colon and/or rumen samples. In a third trial that inoculated eight calves with a four-strain cocktail of O157:H7 strains, the gall bladders from all eight animals were positive 9 days after inoculation. The colon and rumen samples from these calves were also positive. E. coli O157:H7 isolates recovered from bile samples and subtyped by pulsed field gel electrophoresis found that three of the four inoculation strains were present in one or more of the calves. Thus, residence in the gall bladder is not restricted to a single strain. Additional evidence of the ability to localize in the gall bladder of cattle was provided by testing the bile from 150 gall bladders (five collection dates, 30 samples each) obtained at an abbatoir and the isolation of E. coli O157:H7 from four samples (2.7%). This study establishes that E. coli O157:H7 can reside transiently or permanently at a low level in the gall bladder of cattle.