Petri dish bioassays

Various bioassay methods have been used to detect the "natural" release of allelopathic agents. Sane authors preferred, after partial purification, to assay the extracts by petri dish methods for gemination, growth of roots or shoots and other symptoms of seedlings. The bioassays also included tests in soil or sand and also in nutrient solution (Table 3). 

Root elongation bloassay of root exudates. Five ml aliquots of the root exudates were pipetted onto three layers of Anchor1 germination paper In a 10 by 10 by 1.5 cm plastic petri dish. Twenty five radish or tomato seeds were placed in a 5x5 array in each petri dish. Radish seeds were incubated at 20C for 96 hours tomato seeds were incubated at 20C for 168 hours, before the root length was measured. Experimental design was a completely randomized design with three replications (dishes) per treatment per bioassay seed species. The bioassay was repeated each week for 23 weeks. 

Bioassay on Solid Medium. A-9, a medium previously shown to be favorable for antibiotic production by actinomycetes in shake flasks (36), was modified for bioassays on solid medium. We halved the concentration of components in A-9 and adjusted the pH to 6.9-7.1 with KOH to reduce the possibility of osmotic or toxic effects of medium components themselves on seed germination and seedling growth. The medium was amended with 15 g agar per liter and poured into 10 x 10 x 1.5 cm square plastic petri dishes, about 60 ml per plate. 

Bioassay of Extracts. Extracts tested for the presence of cyclohexi-mide were also bioassayed for phytotoxicity. The extracts were redis-sOlved in acetone, and 0.2 mg in 2 pi was applied to 6-cm-dia disks of filter paper. The extract was distributed on the paper with 0.2 ml of methanol. The disks were dried with warm air, placed in 1.5 x 6 cm petri dishes, and moistened with 1.5 ml distilled water. Ten cress seeds were placed on the paper, and after incubation for 3 d at 28 C radicle length of the seedlings was measured. 

Bioassays are performed under sterile conditions in a laminar flow hood. Tomato seeds are previously washed and disinfected with 1% sodium hypochlorite. Seeds are germinated in the Petri dishes containing the S. deppei aqueous leachate. For control, seeds are germinated in 1% agar. Twelve seeds are placed on each Petri dish and kept in the dark at 27°C in a growth chamber. For enzyme activities, 40-50 Petri dishes are used per treatment. Primary roots (radicles) are excised after 72 h, frozen in liquid nitrogen and kept at -70 °C until use. For root growth response, experiments... 

Compounds 58 and 59 showed phytotoxic effects when tested against seedlings of A. hypochondriacus using a Petri dish bioassay. Compounds 58 [IC50 = 6.57 xM] and 59 [IC50 = 3.86 xM] inhibited radicle growth of this species with a similar potency to 2,2—dichlorophenoxyacetic acid [2,4-D IC50 = 18 xM], which was used as a positive control. 

The most common and simplest procedure is to place a few microliters of the test solution over a small puncture wound on a detached leaf. The puncture wound enhances the access of the toxin to the leaf tissue. The leaf is then placed in a petri dish containing a filter paper saturated with water. The top cover of the plate is sealed with parafilm, and the plate is incubated under controlled light and temperature conditions. Toxin activity is usually indicated by chlorotic, necrotic, or colored spots on the leaf. Other methods for bioassay involving CO2 fixation, or effects on organelles, whole plants, protoplasts, tissue cultures, or plant parts are outlined (, 7). 

Seeds of lettuce (Lactuca sativa L. cv. Roman), cress (Lepidium sativum L. cv. Comun), and onion (Allium cepa L. cv. Valenciana), were obtained from FITO, S.L. (Barcelona, Spain). Seeds of wheat and barley (Hordeum vulgare L) were obtained from Rancho La Merced, Junta de Andalucia, Jerez, Spain. All undersized or damaged seeds were discarded, and the assay seeds were selected for uniformity. Bioassays were carried out in 9 cm 0 plastic Petri dishes, using Whatman 1 filter paper as support. 

Seed germination and seedling growth of radish in petri dishes were designed to evaluate the phytotoxicity of OMW fractions, thirteen polyphenols isolated from RO fraction and a mixture of these compounds. Radish seed (Raphanus sativus L. cv Saxa), collected during 1999, were purchased from Improta Co., Naples. To test the inhibitory effect of OMW fractions and RO, 20 seeds of radish were placed on two layers of filter paper (Whatman No. 1) in Petri dishes (90 mm diameter). The paper was wetted with 4 mL of buffered distilled water (BDW) with MES (2-N-[morpholino]ethanesulfonic acid) 10 mM, or test solution (undiluted fraction and a series of dilutions 1 2, one part of fraction to two parts of DW, 1 6 1 8 1 10 and 1 14). All pH values were adjusted to 6.0 before bioassay with MES. Experiments were made in triplicate. 

The leaf disk method is a conventional type of bioassay in which disks of constant area are cut from leaves of a certain plant and are coated with the extract or pure compound (in solution). These are then presented to insects in a petri dish. After a certain amount of time, the percentage of the leaf that has been consumed is estimated, visually17 or photographically with or without software.18 A leaf that is coated with the solvent only is used as the control. This bioassay can be used with small variations, for example, instead of leaves, an artificial diet containing sucrose, flour, or even calcium alginate19 can be mixed with the compound. 

Small quantities of compounds in natural extracts are often a problem when these need to be evaluated in bioassays. Sometimes there is just not enough of the compound isolated to carry out the usual bioassay.20 Microassays have been developed10 1 to overcome this problem. Typically, a microassay is carried out on a thin-layer chromatography plate with a cellulose layer. A small droplet (1.5 pi) of the tested compound in a solvent (1-102 nmol cm-2) is then added on the plate. After the solvent has evaporated a small amount (5 pi) of sucrose solution lmoll-1 is added to the place where the compound was added. In the control the same procedure was followed on a different plate, but with the solvent alone, with no compound added. These two plates are then placed in a petri dish with the test insect species. In the past, when paper chromatography was widely used, a crude plant extract was placed on the origin of the paper and then eluted into bands. The paper was freed of solvent, sprayed with sugar solution, and used directly in a bioassay to see which parts of the paper were not eaten, and therefore of interest for further examination. 

Twenty newly emerged boll weevils were placed in 14 x 2 cm petri dishes containing the test and control plugs. The bioassay was carried out in the dark at 80°F for 4 hours, after which the papers were removed and the punctures counted. 

Dicofol is relatively fast acting and, therefore, amenable to use in 24 h, rapid bioassays (21), whereby plastic petri dishes are treated with a discriminating concentration of dicofol and used to estimate the proportion of resistant individuals in populations. 

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