Evaluation of an alternative small stem assay for blight resistance in American, Chinese, and hybrid chestnuts (Castanea spp.)

We tested an alternative small stem assay (SSA) for blight resistance in chestnuts (Castanea spp.). Whereas standard SSAs are done by inoculating small incisions in stems, we cut off stems (4 to 5 mm diameter), inoculated the cut ends with disks of Cryphonectria parasitica inoculum, and covered them with plastic sleeves. This method was designed to be relatively simple to implement, to consistently induce cankering, and to better enable seedlings to recover by developing shoots from the lower stem (standard SSAs delay removal of blighted stems until late in the growing season, if at all). We conducted six experiments with seedlings and orchard trees of Castanea dentata (susceptible), C. mollissima (resistant), and hybrids expected to vary in resistance. Experiments with seedlings and two of the three orchard experiments showed clear differentiation between susceptible and resistant types, especially after 90 days post-inoculation and when the orange-colored zone of the canker was measured. One orchard experiment failed to give clear results, but was ended earlier (60 days) than the other experiments. We observed only two failed inoculations out of over 200 performed. Comparisons with other studies suggest that this SSA method performs at least as well as the standard SSA method in distinguishing resistant and susceptible types, at least in seedlings. Survivorship after one year for seedlings inoculated in 2018 ranged from 70% for C. dentata to 100% for C. mollissima, and in 2019 ranged from 40% in F1s to 100% for C. mollissima. Deaths of seedlings following SSAs were mostly unrelated to the inoculations (e.g., root rot).

Application of an optimized immunostaining technique to evaluate the heterogeneous secretory response from porcine somatotropes by cell blotting.

The objective of the present study was to quantify the absolute hormone release from individual porcine pituitary cells incubated on polyvinylidene difluoride (PVDF) transfer membranes (cell-blot assay). After immunoperoxidase staining, growth hormone (GH) release from isolated somatotrope cells appeared like a colored zone of secretion surrounding the cell. Optical densities of these secretion zones were quantitated by computerized image analysis and translated into picograms by means of an appropriate standard curve. As a prior step, the staining method and the optimal immunocytochemical conditions were selected by applying purified porcine growth hormone (pGH) to the transfer membranes. The avidin-biotin-peroxidase nickel-intensified (ABC-Ni) method produced a better resolution than the peroxide-anti-peroxidase (PAP) method, although both techniques were similar with regard to background, sensitivity, and range of quantitation. The amount of GH released from single porcine somatotropes was highly heterogeneous, although the cells were treated under the same conditions. Moreover, this fact was consistent with the stimulation of the average release of GH by GH-releasing factor (GHRF) but not by GHRF+somatostatin (SRIF). Our results confirm the availability of the recently developed cell-blot assay and support the concept of functional heterogeneity in anterior pituitary cell populations.

Enzyme immunochromatography--a quantitative immunoassay requiring no instrumentation.

We describe a novel test-strip immunoassay for quantifying drugs in biological fluids. This enzyme immunochromatographic ("immunograph") method combines many features of the enzyme-channeling homogeneous immunoassay with immunochromatography and capillary migration to provide a non-separation, non-instrumental assay for theophylline in which quantification is based on the spatial distribution of enzyme label rather than on the modulation of enzyme activity. Sample antigen and hapten-enzyme conjugate are combined and moved by capillary action up a paper strip on which specific antibody has been immobilized. After color development, the assay result is evaluated by measuring the height of the colored zone on the test strip. Quantification is not a function of enzyme activity, so the method is relatively insensitive to sample matrix effects, enzyme instability, temperature, and incubation timing. Either whole blood or plasma can be used as sample. Results correlate well with those by established instrumental methods. The simple, rapid (15 min), two-incubation protocol is well suited for on-site testing in non-laboratory environments.

Inhibition of Psychrotrophic Bacteria in Refrigerated Milk by Lactobacilli Isolated from Yogurt1

A “milk-peroxidase” agar was prepared to select cultures of lactobacilli that most actively produce hydrogen peroxide. O-tolidine and peroxidase contained in the medium reacted with hydrogen peroxide produced by the bacteria to form a colored zone around the colonies during their formation on the agar at 37 C. There appeared to be no relationship between the size of zones surrounding the colonies on the agar medium and the amount of peroxide produced in milk at 5.5 C. Cells from frozen concentrated cultures of selected lactobacilli were added to milk to achieve desired populations in the interaction experiments. A significant inhibition of a psychrotrophic culture in autoclaved milk at 5.5 C occurred when at least 2.5 × 107 lactobacilli were added per ml. Larger numbers of lactobacilli resulted in greater inhibition of the psychrotroph. The intensity of inhibition of a psychrotrophic culture in autoclaved milk varied among the cultures of lactobacilli tested. In most instances, the more active inhibitory cultures produced more peroxide in refrigerated milk than less inhibitory ones. In preliminary experiments, a culture of Lactobacillus bulgaricus selected by the above procedure produced no inhibitory effect against the growth of psychrotrophic bacteria in non-agitated raw milk at 5.5 C.