RESEARCH
Enzyme Activity Causes Plant Wilt Wisconsin biochemists find wilt-producing fungal enzymes are less active in resistant plants than in susceptible ones
TOMATO WILT. Dr. Dawson C. Deese and Dr. Mark A. Stahmann examine a tomato plant for signs of wilt. Fusarium fungus, for instance, wilts the plant's leaves and causes the vascular system to brown; growth is thus stunted and the plant soon dies
Some plants resist wilt diseases while others do not because of differing ac tivity of certain enzymes that cause plants to vviit. Susceptible plants show much more enzyme activity after infection tham do resistant ones, ac cording to Dr. Dawson C. Deese and Dr. Mark Λ. Stahmann of University of Wisconsin. Thus, genetic resist ance in plants may have a biochemical basis. The Wisconsin biochemists reached these conclusions from experi ments with fungi that produce wilt in tomato, banana, and potato plants. Two of the more common fungi are Fusarium and Verticillium. in Fusa rium wilt of tomatoes, for instance, the leaves permanently wilt and the vas 38
C&EN
JAN.
2 3,
1961
cular system browns. As a result, the plant is stunted and usually dies within 30 days. Parasitic fungi such as these grow in the vascular sys tem of plants and produce wilt dis eases that cause annual losses amount ing to millions of dollars. In attacking a plant, Fusarium fun gus enters through the roots and passes into the vascular system. There, it produces enzymes, among which are pectic depolymerase and pectin methyl esterase. These en zymes, in turn, break down some of the insoluble pectin and form pectic gels in the vascular elements. The gels block water transport and cause the plant to wilt.
Of these two enzymes, pectic de polymerase appears to be the major culprit, according to earlier work at the university (C&EX, April 21, 1958, page 47 J. Pectin methyl esterase it self causes little wilt. "In carrying on this work, we decided to study the role of depolymerase in Fusarium wilt," Dr. Deese says. First, he grew the fungus on stem tissue obtained from both resistant and susceptible tomato varieties. Fungus growth proved to be about the same on both. After five days, he extracted these cultures with water and cheeked the pectic enzymes formed by the fun gus, lie found that susceptible stem sections contain three to tour times more depoK merasc than resistant ones do. Dr. Deese then repeated the experi ment on Fusarimn-iiifeeted, intact plants. lie inoculated tomato cuttings of susceptible and resistant plants with fungus spores and grew new roots on the plants. After two weeks, the bio chemist harvested both infected and noninfected tomato stems from the plants, then washed and froze them. Next, he thawed the stems and ana lyzed their juice for pectic enzymes. "We found that depolymerase activity rose 300'^ in susceptible stems; there was no increase in pectic enzymes in the resistant plants. Dr. Deese says. Thus, he concludes, pectic depoly merase must play a decisive role in Fusarium wilt.
Action in Bananas and Potatoes. The Wisconsin research workers also studied fungus growth and depoly merase activity in banana and potato plants. The organism used, Fusarium oxi/s})oriuin /. rubciisc, is the4 fungus that affects the banana and causes Panama disease. Dr. Deese explains. In one experiment, he grew the fungus on pseudostem tissues from two susceptible and four resistant plants grown in the greenhouse. F. cubense produces two to three times more pec tic depolymerase on tissue from the two susceptible varieties of banana
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.
2 3, 1 9 6 1
C&EN
39
