Direct inoculation assay

Detached leaf assays provide us with the opportunity to evaluate new fungicides directly on the leaf surface in a dose-response format (Table 1). This assay allowed us to benchmark potential lead compounds such as CAY-1 and sampangine with a commercial standard (azoxystrobin) of known mode of action (Qo I inhibitor). The number of diseased lesions was used to determine effective concentrations needed for disease control. Lesion size is used to determine the relative effectiveness of the systemic activity that produced curative activity 24 hrs after inoculation. The detached leaf assay was also used to establish experimental field rates for future studies. Study of protectant activity indicated that 1250 ppm. CAY-1 or sampangine appeared to be an effective concentration for disease control of anthracnose on the leaf surface, or between 100-1000 times the concentration required for in vitro activity (Post, Table 1). 

Host-mediated assay, m the host-mediated assay, target cells cure inoculated into an animal that receives the chemical treatment. The assay can be performed only with cell lines that do not kill and cure not killed by the host. Several chemicals—including AF-2, EMS, DMN, DEN, and MNNG—have been tested in this assay system. These chemicals induce mutations in the direct test or in cell-or microsome-mediated assays. Such studies may be useful in understanding responses—including tissue distribution, activation, detoxification, and elimination of chemicals— in whole animals. 

In this series of bioassay experiments, several carcinogens failed to transform certain batches of hamster embryo cells, presumably because of a lack of endogenous enzymes required for the metabolic activation of these compounds. In short-term bacterial and mammalian cell mutagenesis systems, these enzymes are routinely provided by the addition of rat liver homogenates. As mentioned earlier, diethylnitrosamine and urethane, which failed to transform hamster embryo cells directly, were activated in a host-mediated in vivo-in vitro cell transformation assay in which the chemicals were inoculated intraperitoneally. In our laboratory, when A -2-acetylaminofluorene, 4-aminoazobenzene, auramine, diethylnitrosamine, 3-methoxy-4-aminoazoben-zene, Natulan, 2-nitrofluorene, p-rosaniline, and urethane were tested in the presence of hamster liver homogenates and appropriate cofactors, all except 4-aminoazobenzene gave positive results. The liver homogenates were prepared from hamsters that were not treated with enzyme inducers such as Aroclor 1254 or phenobarbital. 

Another reason for careful interpretation of the disk-diffusion assay is that it is subject to false-positive results that can be misinterpreted as antibiotic activity. For example, physical characteristics of the extract (viscosity, pH, etc.) can generate small zones of growth inhibition when bacteria are inoculated directly onto the surface of the agar plate. In addition, we have observed that some primary metabolites can inhibit growth when tested at high concentrations. It is also possible that simple molecules, or extract degradation products, can exhibit mild antibiotic properties. For these reasons, it is important that replicate extracts are tested and that small zones of inhibition are interpreted with caution. It is also important to clearly state the concentrations tested, even if naturally occurring concentrations are not known, so that activities can be reproduced and evaluated at a later time. 

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