Thesis (B.S.)--University of Illinois, 1920.Typescript.Includes bibliographical references (leaf 15)

Budhe, Janardan Sahasra

English

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The production of injury to bean leaves by certain arsenicals

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tezoB85J. 5. Bud heThe Production of Xnjuru, to BeansLea\re s bu C&rfa'm A r5en icq i 5)THE PRODUCTION OF INJURY TO BEAN LEAVESBY CERTAIN ARSENICALSBYJANARDAN SAHASRA BUDHETHESISFOR THEDEGREE OF BACHELOR OF SCIENCEINAGRICULTURECOLLEGE OF AGRICULTUREUNIVERSITY OF ILLINOIS1920UNIVERSITY OF ILLINOISJune..3, i9gQ...THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION BYJanardan Sahasra BudheENTITLED ProdU^.ArseniealsIS APPROVED BY ME AS FULFILLING THIS PART OF THE REQUIREMENTS FOR THEdegree OF... ...J&sMl©X...©.£...ScienceApproved :HEAD OF DEPARTMENT OF .....H.Ortic.Ul tur e.Digitized by the Internet Archivein 2013fhttp ://arch ive .org/deta^Table of ContentsI MaterialsII — ExperimentalHI ResultsIY Character of InjuryV DiscussionVI ConclusionsVII BibliographyThe Production of Injury to Bean Leavesby Certain ArsenicalsIt has long sinoe been proved that arsenical sprays in-jure the foliage leaves of many plants of economic importance. (5)The following pages contain the results obtained by experiments withsodium arsenite , calcium hydrogen arsenate and tri-calcium arsenate,applied to bean leaves. The object was to investigate, to someextent at least, the degree and kinds of injury produced by thesethree sprays, and the conditions governing its occurrence.The writer is greatly indebted to Doctor W. A* Ruth forhis kind advice and valuable suggestions, and wishes to express hishearty appreciation for his help and kindness.I MaterialsThere are several methods by which sodium arsenite,Na^AsOg, can be prepared. The following method was used because ofits simplicity. Twenty-five grams of pure anhydrous sodium car-bonate, RagCOg, were dissolved in £50 cc. of water in a 500 cc.measuring flask, and to it was added 4.95 grams of Baker and Adam-son^ CP. arsenic trioxide, ASgO^, and it was heated on the waterbath until all of the arsenic trioxide was dissolved. It was thenzcooled to room temperature. The resulting solution was alkalinebecause of the large excess of sodium carbonate used. Therefore,it was made neutral to litmus with hydrochloric acid, HC1, so thatthe effect, when used as a spray, would not be complicated by strongalkalinity.Robinson's (8) procedure was followed for the preparationof calcium hydrogen arsenate, CaHAsO^. He has shown that the saltis formed by the reaction between calcium chloride, CaClg, and sodi-um hydrogen arsenate, Ha HAsO , according to the following equation:CaClg plus Na2HAs04equals CaHAs04plus 2 HaCl. Solu-tions of calcium chloride and sodium hydrogen arsenate were preparedby dissolving 15 grams and 18.6 grams of each respectively. Thisgives an excess of calcium chloride over the amount required in theequation above. The solutions were slightly acidified with hydro-chloric acid. After filtering, they were diluted to 250 cc. each.Then the cold solution of calcium chloride was slowly added to thecold solution of sodium hydrogen arsenate with constant stirring.A white precipitate formed which settled slowly. The clear, super-natant liquid was decanted and the precipitate washed further by de-cantation several times. Finally, it was brought upon the Buchnerfunnel and washed there with hot distilled water several times. Itwas then dried at room temperature. The dried salt was a whitepowder•The method given by Robinson (8) for the preparation oftri-calcium arsenate, CagfAsO^Jg from calcium chloride, sodium hy-drogen arsenate and sodium hydroxide was tried without success,3because of the formation of a colloidal precipitate tinder many vary-ing conditions of concentration and temperatures* It was foundthat a powdery precipitate was formed when lime water was substitutedfor calcium chloride. Calcium hydroxide, Ca(0H) 2 , was prepared byslaking 20 grams of lime with a little water, diluting to 4000 cc.and filtering after standing for half an hour, Merck 1 s CP. sodiumhydrogen arsenate was dissolved in water and just enough of a solu-tion of sodium hydroxide, NaOH, was added to make it neutral to lit-mus. It was then filtered to get a clear solution. The lime waterwas slowly added to the clear solution of sodium arsenate with con-stant stirring. Immediately a voluminous precipitate was formedwhich remained in suspension. Upon heating to 90 degrees C. theprecipitate settled very rapidly. The supernatant liquid was de-canted and the precipitate was further washed several times by de-cantation with hot distilled water to get it free from sodium hy-droxide (litmus). The precipitate was dried at room temperatureand a white, apparently amorphous, salt was obtained.Bush Bean (Phaseolus vulgaris L. ) seed of the variety ExtraEarly Refugee was used. This was obtained in March, 19£0, fromVaughan's Seed Store, Chicago, Illinois. The seeds were planted inrows, four rows in a flat and six seeds in a row. The seeds wereplanted on the twenty-sixth of March. The flats were watered when-ever it was necessary.The flats used were fifteen by twenty-two by three and one-half inches, inside dimensions. The soil used in the flats wasprepared by thoroly mixing together four parts of ordinary gardenFigure I« Arrangement of Treatments and Cheeks5Sodium arsenite spray1 C 2 CA B A BU.M. U.M. U.E. U.E.3 C 4 C CA B A BL.M. L.M. L.E. L.E.Calcium hydrogen arsenate spray1*C f3 2 C CA B A BU.M. U.M. U.E. U.E.3 C C 4 C CA B A BL.M. L.M. L.E. L.E .Tri-calcium arsenate spray1 C C 2 C CA B A BU.M. U.M. U.E. U.E.3 G G 4 C cA B A BL.M. L.M. L.E. L.E.Explanation: - Humber is the number of the flatC means CheckA means strength of solution - 2 lbs. to 50 gallonsB means strength of solution - 1 lb. to 50 gallonsU.M. - only upper side of leaf sprayed in morningU.E. - only upper side of leaf sprayed in eveningL.M. - only lower side of leaf sprayed in morningL.E. - only lower side of leaf sprayed in evening6The young plants began to appear above the ground by thethirty-first of March. At the end of one week about 75 percent ofthe seed was up* No plants were observed to come up after thisdate,II ExperimentalArrangement of treatments and checks was made in the man-ner shown in Figure I. The entire number of flats used was twelve.These were divided into three sets of four flats each. One set wasfor each of the three materials. Each flat was divided into foursections. Plants in two sections on one side and adjacent to eachother were used for checks; and those in the remaining two sectionsof the same flat were used for spraying. The purpose in doing thiswas to insure uniform conditions of temperature, light and moistureto both the checks and the treatments.Suspensions of the arsenates were prepared beforehand andwere allowed to stand for three days before the first application.Only the supernatant liquid from the calcium arsenates was used inspraying. Suspensions were made of two different concentrations.One was made by suspending the material at the rate of two pounds tofifty gallons and the other was made similarly with one pound tofifty gallons. One pound to fifty gallons is the standard spray ingeneral use. Sprays were applied with a DeVilbiss atomizer.Spraying was done in the mornings and evenings to seewhether the differing external conditions of light, heat and humiditytaken as a whole, caused variations in effect.Upper and lower surfaces of the leaves were sprayed in or-der to determine which of the two surfaces was more susceptible toarsenical injury under the same conditions of temperature, light andmoisture.Dark green leaves and pale green leaves were sprayed anddusted with powders of calcium hydrogen arsenate and tri-calciumarsenate. The purpose in doing this was to see whether the leaveshaving more chlorophyll were more or less susceptible to the arsen-ical injury than those having less chlorophyll. It has been provedconclusively that dark green leaves contain much more chlorophyllthan pale green leaves (4).Upper and lower surfaces of leaves were dusted with pow-ders of calcium hydrogen arsenate and tri-calcium arsenate, theleaves being kept dry. The object of doing this was to ascertainwhether water was necessary for the production of injury. It ispresumed that only arsenicals in solution cause injury (8) and bycertain investigators that the carbon dioxide and water given off inrespiration is a factor in the production of injury.The first application was made on the sixth of April whenthe leaves were about five to seven cc. long. This was followed bytwo more applications on the same leaves, one on the seventh and theother on the eighth of April. The sodium arsenite and calcium hy-drogen arsenate sprayings were discontinued after the third applica-tion because of the fact that the leaves sprayed with the formerwere totally destroyed by this time and those sprayed with the8latter had 40 percent of their surfaces injured. Spraying withtri-calcium arsenate was continued for five days. At the end ofthis period very little of the foliage was injured.Ill Be suits1* Leaves sprayed with sodium arsenite were completelydestroyed "by three applications. Ho difference in the severity andextent of injury was noticed "between the leaves which were sprayedon their upper and lower surfaces, or between those sprayed in theevening and those sprayed in the morning.2. Three applications of calcium hydrogen arsenite sprayinjured, on the average, 40 percent of the leaf surface.3. Ho leaves sprayed with tri-calcium arsenate were in-jured as a result of five applications. After the fifth spray onlya few spots were noticed, most of them being on leaves sprayed onthe lower surface.4. jDeaves which were sprayed on the under surface wereinjured more than those sprayed on the upper surface.5. Dark green leaves were less injured than pale greenleaves.6. A very little injury was produced by dusting of cal-cium hydrogen arsenate on lower and upper surfaces of leaves.7. No effect whatsoever was noticed from dusting withtri-calcium arsenate.8. The leaves which were sprayed in the mornings and71those which were sprayed in the evening showed no difference in theseverity and intensity of injury produced.Table I. - Comparative Injury Produced by Applications ofCalcium Hydrogen Arsenate and Tri-Calcium Arsenate on Upper and lowerSides of LeavesEstimation of injury was made by taking the whole leaf surface as100 and the injury is expressed here in percentage. For example,if the injury is stated to be 10, it means that 10 percent of thefoliage surface was destroyed.Spray ma-terialusedStrength ofSpray so-lutionInjury pro-duced byspraying onupper sur-faceInjury pro-duced byspraying onlower sur-faceAveragepercentof in-juryNumber ofapplica-tionsCaHAsO,CaHAsO.Ca3 (As04Ca3 (As04 )2£# to 50 gall1# to 50 "to 50 «1# to 50 »)22#3020.01.0015040.1.0054030.055.0033355The table given above shows that calcium hydrogen arsenateproduced very much more injury than tri-calcium arsenate; and thatthe injury to leaves which were sprayed on the lower side is con-siderably more than the injury to leaves which were sprayed on theupper surface. In other words, the lower surface of bean leaves ismore susceptible to calcium arsenate spray injury than the upper sur-face of bean leaves.10IV Character of InjuryInjury appears to occur anywhere on the leaf, The in-jured tissue "before it dried, was lighter by reflected, but darkerby transmitted, light. It soon dried and the color of the driedtissue when held against the light still was dark. Freehand micro-scopic sections of freshly injured tissue were made by cutting inelder pith. Under the microscope the injured tissue appeared black.Upon closer examination it was seen that the chloroplasts were dis-sociated. The section on the slide was heated with a drop of waterto see whether the dark color was due to the presence of air, butthe dark pigment still remained. It was then heated with 95 per-cent alcohol to see whether the dark pigment resembled chlorophyllin being soluble in it. But the dark pigment still persisted asbefore and the alcohol did not becomB green. Therefore, it appearsthat a new dark pigment was produced at the expense of the chloro-phyll •V DiscussionThe results stated in the foregoing pages can now be dis-cussed. The weather record shows that the weather during theThe weather conditions:- April 6, fair and warm; April 7, fair andwarm; April 8, fair and warm in the morning and at noon but cloudyand warm in the afternoon and evening.11first two days when the sprays were "being applied, was fair andThe weather conditions during night times were different fromthose during day times; the conditions with respect to temperatureand humidity, at least, were, however, more or less uniform in thegreenhouse during night times.warm* Change in the weather came in the afternoon of the thirdday, when it was warm and cloudy. The sodium arsenite and calciumhydrogen arsenate, put on during these three days only, produced in-jury independent in severity and extent of the weather. Injuriesproduced "by the sprays applied in the mornings and in the eveningsdid not differ in any way as regarded severity and amount. Again,on the fourth and fifth days there was a sudden drop in the atmos-pheric temperature and it was cloudy. During these two days thetri-calcium arsenate produced a little injury, while it had producedno injury during the first three days when the weather was fair andwarm. It seems probable that the difference is to be attributed tothe slower drying of the spray during the cold, cloudy weather althono notes were made of the rate at which the spots of spray driedunder the two conditions of weather.The results show that the lower surface of the bean leafis more susceptible to arsenical injury than the upper. It hasbeen proved that "all plants have the power of absorbing water thrutheir leaves" (6). Burgerstein (3) says that "in general, theplants in which the absorption of water by leaves has been shown re-veal a wide range in organization, mode of life and systematic12position. Therefore, since no exception has been shown it may beassumed that all plants have the power of absorbing water directlythru their leaves". Discussing the means by which the leaves ab-sorb water he further observes that "the water can reach the inter-ior of the leaf thru the epidermal cells, thru the hairs, and thruthe stomata. The lower epidermis absorbs more actively than theupper as investigations have thus far shown. As a rule threefactors combine to cause the greater absorption by the lower side ofthe leaf; (a) The fainter cuticularization of the outer walls ofthe epidermal cells; (b) the greater abundance of hairs; and (c) thegreater numbers of stomata" (1). The bean leaves under theHaberlandt, (6) asserts that under no circumstances do stomata ab-sorb water.microscope showed more stomata and hairs on the lower surface thanon the upper. It then appears that the greater susceptibility ofthe lower surface of the bean leaves is directly related to the pre-sence of a greater number of stomata and hairs on it. It may bequestioned, however, as to the possibility of absorbing arsenicalsolutions. This has been proved conclusively by Uoussingault (2)who says "that not only leaves may thus take up water but also atthe same time will take up saline substances in solution".The greater susceptibility of pale green leaves to thearsenical injury may be indirectly associated with the fact thatpale green leaves contain a smaller amount of chlorophyll than the13dark green leaves (4). The smaller the amount of the chlorophyll,the less is the activity of the chloroplasts and the protoplasts ingeneral; and a change in the susceptibility of the protoplast may beanticipated, A difference in the structure of the tissues may alsobe a factor, e.g., of the epidermis, cuticle, and hairs.Dusting with calcium hydrogen arsenate, the leaves beingkept dry, produced very little injury. Gillette (5) found the samething with arsenites. But a contrary view is shared by Patten andO fMara (7) who say that most of the injury that occurs from arse-nates is due to the dissolving of arsenates by the carbon dioxideand water liberated in respiration. All plants give off water andcarbon dioxide at night in the process of respiration. The waterthus given off is charged with the carbon dioxide and forms a thinfilm on the surface of the leaf. Patten and O'Mara have, indeed,proved that carbonated water dissolves arsenates much more thanordinary water, but this explanation, it seems from the author T s ex-periments, does not hold. Tri-calcium arsenate, which is veryslightly soluble in water and is much more soluble in carbonatedwater, produced no injury whatsoever when applied in the dry form tothe bean leaves. Injury, therefore, must have been initiated im-mediately after the wet material is applied, while liquid water wasstill present. Neither does the evolution of carbon dioxide inrespiration seem to be an important factor in the production of in-jury since no more injury occurred following applications made justbefore it became dark in the evening than followed early morningapplications.14VI Conclusions1* Sodium arsenite produces the most injury to beanleaves,2, Tri-calcium arsenate produces the less injury to beanleaves than calcium hydrogen arsenate*3. The lower surface of bean leaves is more susceptibleto arsenate injury than the upper,4, Injury is initiated immediately after the material isapplied.5. Injury depends upon liquid water, applied in this casewith the arsenical,6, Respired water is not enough to initiate injury.7. The arsenate injury to bean leaves causes the dis-sociation of chloroplasts and the formation of a black pigment whichis soluble in water and alcohol.15Bibliography1. Bain, Samuel M. t The Action of Copper onleaves, Tenn. Agr. Exp. Sta. Bui. Vol. 15,No. 2, 1902.2. Boussingault,J., Etude sur les fonctionsphysiques des feuilles, Ann. de Chimie etde Physique, Ser. V, Vol. 13, 1873, pp.289-394. (Cited by Bain, S. M.)3. Burgestein, A., Ubersicht der Untersuchun-gen fiber die Wasseraufnahme der Pflanzendurch die OberplSche des Blotter, Jahresb.Leopoldst. Comm-Real-u Obergymnasium WienXXII, 1891, pp. 3-47. (Cited by Bain, S.M.)4. Curtis, C. C, Uature and Development ofPlants, H.Y. , 1916.5. Gillette, C. P., Experiments with Arsenites,la. Agr. Exp. Sta. Bui. 10, 1890.6. Haberlandt, G.,Physiological Plant Anatomy,English Edition, London, 1914.7. Patten, Andrew J., and O'Mara, P., CalciumArsenate, Causes of Injury, Mich. Agr. Exp.Sta. Quarterly Bui. Vol. 2, Mo. 2, pp. 83-84.8. Robinson, R. E. , Calcium Arsenate, Oregon AgrExp. Sta. Bui. 131, 1918.UNIVeRSfTY Of ILUNOt«-OBBAN»3 0112 086765333

Typescript.Thesis (B.S.)--University of Illinois, 1920.Includes bibliographical references (leaf 15).Mode of access: Internet

Budhe, Janardan Sahasra.

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