than on comparable tissue from two of the resistant varieties, the biochemists find. The other two resistant species do not form depolymerase. These results point to two things, Dr. Deese says. One, resistant banana plants (like resistant tomato plants) must contain some factor which sup presses pectic enzyme formation. Thus, a study of growth and enzyme activity of the fungus could shorten an assay for resistance in banana plants from several years to a few months or even days, he feels. Second, the fun gus causing Panama disease closely re sembles the one that makes tomatoes wilt. This could mean that vascular plugging in the banana plant may also come from pectic depolymerase of the pathogen. Another fungus, Verticillium alboatruni, attacks plants in much the same way as Fusarium does, although it af fects both tomatoes and potatoes. In studying this organism, the biochem ists used two isolates—one from to mato, the other from potato—to in oculate tomato stem tissue of the susceptible plant (Bonny Best variety) and two resistant ones—Loran Blood and VR Moscow. "We find that in Bonny Best, the Verticillium fungus produces three to 10 times more pec tic polygalacturonase than it does in Loran Blood. But in the highly re sistant YR Moscow7, it shows no en zyme formation," Dr. Deese says. The biochemists also studied a Ver ticillium isolate which attacks only potato plants. They infected the plants by immersing their roots in a spore suspension of the fungus. Dis ease symptoms, including stunting, appeared as early as 21 days in sus ceptible plants and increased steadily; resistant plants displayed few or no symptoms. Correspondingly, large amounts of pectic polygalacturonase formed in the susceptible plants, while the resistant ones showed no increase in this enzyme. Summing it all up, the Wisconsin team says that plants in which Fusarium or Verticillium can make enough pectin-splitting enzymes are suscepti ble, and develop disease symptoms. Resistant varieties, though, suppress or inhibit these enzymes. "Our results point out that this is the biochemical way that the genetic resistance in these plants is expressed. We are now studying reactive oxidases and other oxidizing substances that may be re sponsible for suppressing the harmful hvdrolvtic enzvmes," Dr. Deese adds.
TOXICITY. New bioassay for insecticide residues is specific for Phosdrin. Here, Dr. Yun-Pei Sun translates the insecticide's toxicity to pomace flies into residue concentration
Specific Bioassay Picks Out Phosdrin Variations of Shell Development technique may make it useful for determining other pesticides A bioassay for pesticide residues which is highly specific for Phosdrin insecticide ( 1-methoxyearbonyl-l-propen-2-yl dimethyl phosphate) has been developed by a research team at Shell Development, Modesto, Calif. The new method is based on the in secticide's toxicity to an organism, and translating this toxicity into concentra tion of the insecticide. The bioassay was developed by Dr. Yun-Pei Sun, Dr. John San jean, and D. M. DeVries. Bioassays are widely used in the early stages of insecticide development before chemical assays are available, but they are generally considered to be of limited utility because they are not specific. And Food and Drug Admin istration requires a specific method for residue determination before a tolerance for a new insecticide can be set. Now, the Shell Development scien tists have worked out a bioassay which they say is not only specific, but is accurate and sensitive as well. Dr. Sun believes that the principles in volved should lead to the development of bioassays that are specific for other insecticides as well. The specificity of the method, when tested in the presence of 11 other cholinesterase-inhibiting organophosphates, is such that in no case is the interference more than lc/c. Anything
up to 1 0 ^ interference is usually con sidered to be satisfactory in a specific analytical method for residue deter mination. To achieve this degree of specificity, Dr. Sun relies on three characteristics of Phosdrin: • High toxicity to the test organism. • Quick action. • Low masking effect of plant ex tractives on the toxicity. Procedure. Dr. Sun cuts a sample of Phosdrin-treated crop into small pieces and extracts it with chloroform in a homogenizer. After filtering and drying (over sodium sulfate) the ex tract, which now7 contains the insecti cide, he uses it directly for the determination. He places 1 ml. of a 0.02'ί solu tion of peanut oil in chloroform into a jar, adds 2 ml. of the extract, and evaporates the solution to dryness with gentle swirling on a shaking table. This leaves a thin film of toxi cant on the bottom of the jar. He then puts 50, one-day-old pomace flies (Drosophila melanogaster) in the jar along with a pad of cotton soaked in apple juice for food, and covers the jar with a piece of thin paper. After four hours' exposure to the residue, the flies are transferred to a counting cage and the dead and moribund ones counted. JAN.
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Standard Series. For each anal ysis, Dr. Sun also carries through a scries of toxicant-free extracts to which known amounts (from 0.3 to 2.0 micrograms) of Phosdrin have been added. A plot of per cent mor tality against toxicant quantity gives a straight line on log-probability paper. He then uses the mortality count pro duced by the sample to read Phos drin quantity from this chart. The relatively short time of ex posure of the flies and the small quan tity of toxicant used ensure that other organophosphates that may be pres ent will have little effect. Then, too, the toxicity of other organophosphates is masked by the fruit or vegetable extracts to a considerably greater ex tent than is that of Phosdrin. This simple procedure with no cleanup of the extract permits de termining as little as 1 p.p.m. of Phos drin. By using a cleanup procedure involving adsorption on and elution from a carbon column, followed by liquid-liquid partition (Phosdrin is highly water soluble, unlike other organophosphate insecticides), specific ity can 1)2 further improved and sensi tivity can be extended down to 0.1 p.p.in. For example, celery extract containing 0.3 p.p.m. Phosdrin and 3.0 p.p.m. Diazinon [0,0-diethyl-0(2-isopropyl-4 - methyl - 6 - pyrimidyl) thiophosphate] gives a bioassay result of 0.29 p.p.in. Phosdrin after cleanup. Generally Applicable. Dr. Sun be lieves that developing bioassays that are spécifie for other insecticides can be done in several ways. By taking advantage of the different physiological responses of insects, many possibilities for increasing specificity can be introduced. And adding one or more chemical cleanup steps can increase specificity still further, he says. Bioassays will become increasingly important, Dr. Sun feels, as more insecticides are developed. With many of these being chemically similar, it will be increasingly difficult to devise highly specific chemical procedures. But chemically similar insecticides can have very different effects on insects. And when these differences are properly used, they can lead to sensitive and specific methods for determining very small amounts of pesticide residues. Also, plant extractives can interfere with chemical methods based on functional groups. But these extractives are usually nontoxic and would have little effect on a bioassay, | he notes.
