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Some effects of sulphur dioxide on coniferous trees Hughes, Norah Louise 1934

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U . B . C . LIBRARY!  •I ICC". 1 ^ . - J J L ^ ^ b ^ E ^ ^ .  SOME EFFECTS OF SULPHUR DIOXIDE  ON CONIFEROUS TREES  No rah. L o u i s e Hughes, £. A .  A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ARTS IN THE DEPARTMENT of BOTANY  THE UNIVERSITY OF BRITISH COLUMBIA MAY, 1934  j  gable  of  Contents.  Page  Acknowle dgement s.  Statement of the Problem and Scope o f the present study  Review of the L i t e r a t u r e  1  —  •  —  —  M a t e r i a l and Methods i n Experimental work E x p e r i m e n t a l work  Fumigations  — - — — — v  v  t  P  ~ — — — —  6  •  •  — —  -  2  — —  7  ~™  7  M a t e r i a l and Methods •*•»•»•—-—--—  ——-~—•—-  7  Method of a n a l y s i s o f gas samples  —  7  MeMiod of Fumigation General r e s u l t s  —  -  — —  •—•  —  •  —  S u p e r f i c i a l symptoms of i n j u r y  — —  —  —  —  11  —  —  11  —  11  — — — — • — —  S p e c i f i c rate of i n j u r y  — —  —  12  Factors involved i n determining the r a t e — — —  of i n j u r y  —  13  H i s t o l o g i c a l studies  —  19  M a t e r i a l and methods  — - — • — • — —  20  Study of l e a f sections Normal specimens  —-—— - — — —  •  M a t e r i a l and methods  23 •  • •—•  21  —  — • — • — —  Food storage  21  — — - — —  —  Fumigated specimens P h y s i o l o g i c a l studies  •  —• —*  31 31 31  of (W.m^.g.  gable  Acknowledgemsnts. Page  Results  —  —  Discussion o f results  —  Dessication Studies — — — —  M a t e r i a l and mathods —  Results  —  Summary. — —  3  3 44 53  •  53  —.—~—~  54  ——-—-  56  —  Some suggested problems f o r f u r t h e r study a r i s i n g out o f the above experiments  ——  Explanation of plates P l a t e s (1) to (XT) Bibliography.  —•—  57  AOZN'OWLEDGEMEHTS  The w r i t e r w i s h e s to express her deep indebtedness to Dr. A. H. H u t c h i n s o n , Head of the Departnsnt o f Botany a t the U n i v e r s i t y o f B r i t i s h Columbia, f o r h i s u n f a i l i n g i n t e r est i n t h e problem, and f o r h i s many h e l p f u l and encouraging suggestions.  Thanks are a l s o due to Dr. Hutchinson and to  Dr. F. Dickson, A s s o c i a t e P r o f e s s o r a t the U n i v e r s i t y of B r i t i s h Columbia f o r the loan of books and p e r i o d i c a l literature.  Gratitude i s also expressed f o r the k i n d n e s s of  Mr. Garnish a t the U n i v e r s i t y Greenhouse .who cared f o r the specimens used i n the experiments.  SOME STUDIES IK IHE EFFECTS OF SULPHUR DIOXIDE •UPOE  CONIFEROUS TREES  Statement of the Problem and, the Sco-pe of the present  Study.  Much a t t e n t i o n has been drawn to the problem of the i n j u r i o u s e f f e c t s of smelter fumes containing sulphur d i o x i d e upon the vegetation of areas surrounding m e t a l l u r g i c a l workss, and there has been a great d e a l of d i s cussion of t h i s s u b j e c t .  The problem i s a very wide one.  i t i s necessary  to show, f i r s t that any appreciable amount of sulphur dioxide i s given out by the smelter i n question, secondly that the vegetation i s a c t u a l l y i n j u r e d by t h i s sulphur dioxide* t h i r d l y the nature of the i n j u r y , and l a s t l y how i t i s brought about.  The f i r s t p a r t of the problem involves  sampling and analyzing fumes given o f f by the smote staclc.  In order to  demonstrate that the vegetation i s a c t u a l l y Injured by the sulphur dioxide, two l i n e s of i n v e s t i g a t i o n might be followed. By a thorough study of the d i s t r i c t a f f e c t e d i t might be possible to e l i m i n a t e a l l other sources of i n j u r y , or on the other hand, by subjecting p l a n t s to toiown q u a n t i t i e s of sulphur dioxide under c o n t r o l l e d c o n d i t i o n s i n j u r y may be obtained.  This  i n j u r y should be s i m i l a r to that shown i n the area surrounding the smelter.  The t h i r d and f o u r t h l i n e s of i n v e s t i g a t i o n could be c a r r i e d on i n the  f i e l d or i n connection w i t h the c o n t r o l l e d fumigation experiments  and  would include h i s t o l o g i c a l and p h y s i o l o g i c a l research. In the present study no p a r t i c u l a r smelter or area was being i n vestigated so that the f i r s t l i n e of i n v e s t i g a t i o n was not involved and no f i e l d researches were c a r r i e d on.  The l i n e followed was that of  c o n t r o l l e d fumigations w i t h sulphur dioxide*  Stress was l a i d on the  f a c t o r s which a f f e c t e d the degree and rate o f i n j u r y of a l l the species and on the varying rate and degree of i n j u r y shown by the f i v e species under observation.  In an attempt to determine the a c t u a l form of the  i n j u r y , h i s t o l o g i c a l and p h y s i o l o g i c a l i n v e s t i g a t i o n s were c a r r i e d out. The study was l i m i t e d to f i v e species of coniferous trees. Review o f the L i t e r a t u r e There has been a great d e a l of work done on t h i s problem p a r t i c u l a r l y i n the United States of America and i n Germany. Most of the l i t e r ature deals w i t h the i n v e s t i g a t i o n of conditions around m e t a l l u r g i c a l works and i n i n d u s t r i a l areas and the r e s u l t s of c o n t r o l l e d fumigation experiaents. In the United States o f America one of the foremost workers i s J . K. 1  Haywood.  In h i s paper " I n j u r y to Vegetation by Smelter Fumes.  Haywood  gives the r e s u l t s of a s e r i e s of c o n t r o l l e d fumigation experiments.  Work-  ing with Pines he found that a f t e r th£ee, one hour periods in an atmosphere containing one part i n one hundred of sulphur dioxide' the leaves become g r e y i s h and began to droop, while a f t e r s i x hours most of the leaves had f a l l e n and those remaining were y e l l o w i s h brown.  Again, Pines sub-  j e c t e d to three one hour fumigation periods i n an atmosphere c o n t a i n i n g one part of sulphur dioxide In one thousand had some leaves drooping  and  y e l l o w i s h , while a f t e r nine hours a l l the leaves appeared y e l l o w i n g and drooping. 1.  In a concentration of one part of sulphur dioxide i n ten  Haywood J . K., Injury to Vegetation by Smelter Fumes, Dept. of A g r i c u l t u r e , Bureau of Chemistry, B u l l e t i n 89. Government P r i n t i n g O f f i c e , Washington, U. S. A. 1905.  thousand the leaves dropped and became g r e y i s h i n twenty f i v e hours and i n f i f t y hours many leaves became brownish and a l l looked  unhealthy.  Great i n j u r y r e s u l t e d from eighteen hours of fumigation with one part of sulphur dioxide i n t h i r t y thousand.  With cedar two hours i n an atmos-  phere containing one part of sulphur dioxide i n one thousand r e s u l t e d 1 i n leaves becoming brown and drooped.. In another a r t i c l e Haywood  states  that Scrub Pine showed d i s t i n c t i n j u r y a f t e r t h i r t y four hours of fumiga t i o n with one part of sulphur dioxide i n f i f t y thousand, a l l the leaves becoming brown and dry.  N i n e t y - s i x one hour fumigations a l s o a f f e c t e d  a l l the leaves so that the young leaves died and the mature ones were dead at the t i p s .  2  Haywood obtained s i m i l a r r e s u l t s w i t h Cowpeas , and w i t h  3 Black oak, White oak and Chestnut.  Ebough  working w i t h A l f a l f a found  that w i l t i n g r e s u l t e d from two fumigations w i t h one part of sulphur dioxide  i n ten thousand, w i l t i n g and bleaching from three fumigations of that  strength and complete bleaching from seven fumigations.  Four periods i n  ftn atmosphere of one part of sulphur dioxide i n twenty thousand caused w i l t i n g but not bleaching while a l i t t l e bleaching was n o t i c e d a f t e r eight fumigations.  4  Holmes, F r a n k l i n and Gould i n the report o f the Selby  Smelter  1. Haywood, J . K«, Injury to Vegetation and Animal l i f e by Smelter Wastes; U. S. Dept. of A g r i c u l t u r e , Bureau of Chemistry, B u l l e t i n No. 113 (revised). Gov. P r i n g i n % O f f i c e , Washington, U. S. A. 1910. 2. Haywood, J . K. Injury to Vegetation by Smelter Fumes; U. S. Dept. of A g r i c u l t u r e , Bureau of Chemistry, B u l l e t i n No. 89. Government P r i n t ing O f f i c e , Washington, U. S. A. 1905. 3. Ebough, E. W. Gases vs. S o l i d s : An i n v e s t i g a t i o n of the i n j u r i o u s i n gredients of smelter smoke. Journal of the American Chemical Society v 29. Pages 951 - 970. The Chemical P u b l i s h i n g Co. Easton, Pa. 1907. 4. Holmes, J . A., F r a n k l i n , E. C. and Gould, A., Report of the Selby Smelter Commission, U. S. Department of the I n t e r i o n , Bureau of Mines, B u l l e t i n 98., Gov. P r i n t i n g O f f i c e , Washington. U. S. A. 1915.  - 4  -  Commission s t a t e that they found one part i n 1 000 OCO- of sulphur 9  9  dioxide was only s l i g h t l y i n j u r i o u s to barley a f t e r two seven hour fumiga t i o n s and a l s o that s i x seven hour, o r n i n e t y 10 minute fumigations gave 1 l i t t l e effect.  Zimmerman and Crocker  w i t h garden p l a n t s and weeds.  performed a s e r i e s of experiments  They abtained the f o l l o w i n g r e s u l t s .  One  to four hours i n an atmosphere c o n t a i n i n g three o r f o u r p a r t s i n a m i l l i c z i of sulphur dioxide r e s u l t e d i n i n j u r y to such p l a n t s as tomato, coleus, p o t e n t i l i a , nasturtium, chickweed and s a l v i a .  Four to eight p a r t s i n ©  m i l l i o n f o r three to f i v e hours i n j u r e d castor beans, Sorbus Aucupasia, snap dragon,, peach. I l e x apaca, white carnations* gardenias and Bhodaden~ drons were i n j u r e d by f o u r hours i n eight to ten p a r t s p e r m i l l i o n of  2 sulphur d i o x i d e .  Sbaugh  demonstrated that sulphur dioxide i s i n j u r i o u s  to vegetation but does not t h i n k that q u a n t i t i e s s u f f i c i e n t l y large to be harmful would be found near a smelter.  He emphasizes the importance  of f l u dust and suggests that t h i s i s the main f a c t o r i n causing i n j u r y . While not denying the importance of the sulphur d i o x i d e content smelter 3 fumes A. L. Bakke stresses the e f f e c t o f the t a r r y deposit l e f t upon the leaves by smoke and the consequent plugging of the stomata. Holmes,  4 F r a n k l i n and Gould  i n t h e i r report of the Selby Smelter Commission state  that they do not believe the concentration of sulphur d i o x i d e i n the a i r around a smelter t o be harmful i f the out put i s l e s s than f o r t y tons Zimmerman, P. W . and Crocker, W . , Sulphiu* Dioxide i n j u r y to p l a n t s . Proceedings of the Amsrican Society f o r H o r t i c u l t u r a l Science. Vo&. 27 pages 51 to 52. Published by the Society, Geneva, New York, U.S.A 1931. 2. ©p. C i t . 3. BakkB, A. S., The e f f e c t o f Smoke and Gases on Vegetation, C o n t r i b u t i o n of the Botanical Departments Iowa State College, Ames., No. 54. 4. ©p. C i t . 1.  of sulphur a day.  J . V/. Blenkinship who a s s i s t e d i n the work of the Selby 1  Smelter Commission states  i n the report, " I n a l l my t r i p s through the  sm-  oke zone I have only once been able to d i s t i n g u i s h the odor of sulphur dioxide, and then only f o r a few minutes and very f a i n t l y , at a point two and a h a l f m i l e s northeast of the smelter.  Sulphur d i o x i d e to do  m a t e r i a l damage i n dry weather to vegetation i n h a l f an hour must be so strong as to be almost unbearable,  or i f d i l u t e d must be very frequently  repeated or almost constant over the area i n j u r e d i n order to a f f e c t 2 vegetation at a l l . "  Haywood  on the other hand, i n r e p l y i n g to Ebaugh's  a r t i c l e states"...the w r i t e r wishes to e x p l a i n that he i s f i r m l y of the opinion that the s o l i d emanations which a r i s e from a smelter (Including perhaps, soluble copper, a r s e n i c and lead compounds) are i n j u r i o u s to vegetation i n so f a r as they reach i t , but that such emanations reach as f a r as sulphur dioxide or have so i n j u r i o u s an a c t i o n appears to be dec i d e d l y doubtful and c e r t a i n l y has not been proven by the paper published 3 by Professor Chough."  P. Prazer  i n v e s t i g a t e d the causes of i n j u r y to  vegetation i n an urban v i l l a near a large i n d u s t r i a l establishment.  He  found that c l o t h s saturated w i t h a l k a l i n e carbonates absorbed from the atmosphere a large percentage of t h e i r weights, v a r y i n g from 6.06  to  10.85  percent of sulphur t r i o x i d e . He a l s o showed that there was an increased 1. Holmes, J . A., F r a n k l i n , E. C , and Gould, A., Report o f the Selby Smelter Comnission, Dept. of the I n t e r i o r , Bureau of Mines, Government P r i n t i n g O f f i c e , Washington U. S. A. 1315, Page 397. 2. Haywood, J . K. Smelter Smoke. Science V o l . 26., McMillan Co., New York, 1907, page 476. 3. Frazer, P. Search f o r causes of Injury to Vegetation i n an Urban V i l l a near a large I n d u s t r i a l Establishment. Transactions of the American I n s t i t u t e of Mining Engineers. Volumes 38. Published by the I n s t i t u t e New York 1907, pages 498 to 555.  sulphur t r i o x i d e content i n the leaves of t r e e s growing near the i n i.  dustrial plant. of c i t y f o g was  F. W. O l i v e r found that the sulphur dioxide content s u f f i c i e n t to be i n j u r i o u s to greenhouse p l a n t s .  A great deal has been w r i t t e n on some phases of i n j u r y due to sulphur d i o x i d e but t h i s l i t e r a t u r e w i l l (be d e a l t w i t h as each part of the experimental work i s presented. M a t e r i a l and Methods i n Experimental work. The experimental method f o l l o w e d was that of c o n t r o l l e d fumigations with known proportions of sulphur d i o s i d e . species were used i n the present study.  The f o l l o w i n g f i v e  Pseudatsuga t a x i f o l i a .  (Lambefct),  B r i t t o n , (Douglas F i r ) . Pioea s i t o h e n s i s . Oarr. (Sitka Spruce), Tsugfe hetorauhylla, Sarg., (Western Hemlock), Pinus monticola'i Dougl., (Western White Pine) and Thuja t i l i o a t a . Donn., (Western Red Oedar) The trees were four or f i v e years o l d . Specimens f o r immediate use were planted i n ten or twelve i n c h pots and placed under cover i n an open shed, while the remaining trees v/e re planted d i r e c t l y i n the s o i l i n the same shed.  As t r e e s were need-  ed f o r fumigation purposes they were placed i n the greenhouse f o r some time i n order to become a c c l i m a t i z e d to greenhouse c o n d i t i o n s before being subjected to treatment.  Fumigations were made p e r i o d i c a l l y and  material was c o l l e c t e d f o r h i s t o l o g i c a l and p h y s i o l o g i c a l studies.  1.  F. W. O l i v e r . Urban Fogs. Journal of the Royal H o r t i c u l t u r a l Society, Volume (XVI). Spottiswold and Co., London, 1893.  EXPERIMENTAL WORK 1.  Fumigations a.  M a t e r i a l and Methods s  (The fumigations were c a r r i e d out i n a g l a s s sided cabinet having a capacity of approximately 800 l i t r e s .  A l l the j o i n t s between the g l a s s  and the wooden frame were c a r e f u l l y sealed w i t h p u t t y .  The door occupied  the whole of one side of the chamber and could be r a i s e d and lowered at w i l l when p u t t i n g the p l a n t s i n t o the chamber and removing them. Sulphur dioxide was introduced by burning Carbon bisulphide i n alcohol.  Carbon b i s u l p h i d e s u f f i c i e n t to give the necessary  concentration  of sulphur dioxide was added t o a few cubic centimetres of n i n e t y - f i v e per cent me tho 1 a l c o h o l and the mixture i g n i t e d i n the chamber. A small e l e c t r i c fan was used to c i r c u l a t e the a i r and make sura that the  sulphur  dioxide was evenly d i s t r i b u t e d throughout the chamber. In order to f i n d out i f the d e s i r e d concentration vras being abtained, samples of a i r from the cabinet were analyzed. Method of A n a l y s i s o f Gas Samples Apparatus • Two 8 l i t r e a s p i r a t o r b o t t l e s were used f o r taking samples of the gas mixture.  Each was closed a t the top w i t h a rubber stopper f i t t e d w i t h a  s a a l l piece of g l a s s tubing to which a piece of rubber tubing was A pinchoock made i t p o s s i b l e t o close the tube.  attached.  The lower aperture  was  f i t t e d w i t h a one-holed rubber stopper i n t o which a small piece of g l a s s rod was inserted.  Three wide-mouthed g l a s s j a r s , a l l of clear white g l a s s and w i t h a capacity o f about 550 cubic centimeters^  Shsss fcstti&K)were f i t t e d with  rubber stoppers c o n t a i n i n g three holes.  Two of the j a r s ware f i t t e d up as  t e s t i n g j a r s i n the f o l l o w i n g manner.  A piece o f bent tubing having one  arm about two and a h a l f inches long and the other about f i v e inches Icaig was i n s e r t e d w i t h the short end i n the stopper i n such a way that the end of the tubing was j u s t l e v e l w i t h the i n s i d e of the cork. pieces of tubing were i n s e r t e d i n the other two h o l e s .  Two s t r a i g h t  One piece about  three inches long was put i n so that the end was l e v e l w i t h the inner edge of the stopper, the other piece reached to w i t h i n a quarter of an inch of the bottom of the b o t t l e . rubber tubing and pinchcocks.  Both the s t r a i g h t tubes were c l o s e d with  Around the lower part o f each b o t t l e was  pasted a piece of white paper leaving a s t r i p about three quarters of an inch wide through which the colour of the s o l u t i o n in- the j a r could be seen.  One hundred and f i f t y cubic cejil&ffleters of water was c a r e f u l l y  measured into each b o t t l e and the l e v e l marked upon the white paper. t h i r d j a r was f i t t e d up a s a mixing b o t t l e .  The  The f i t t i n g o f t h i s b o t t l e  was the same as f o r the t e s t i n g b o t t l e s except that a small piece o f rubber tubing was attached to the bent glass tubing and that marks f o r one hundred and f i f t y and three hundred cubic centimeters were made on the white paper. In order to be able to discharge the l i q u i d from a burette w e l l inside the t e s t i n g b o t t l e s the burette was f i t t e d with a f i n e drawn out point which would pass through the short s t r a i g h t piece of glass t u b i n g i n s e r t e d i n the rubber stopper.  Solutions needed: An 1J/500 Iodine  Solution  ,1% Starch S o l u t i o n . As i t was needed about 100 ccs. o f 1% starch s o l u t i o n was made up. This was d i l u t e d t o form .1% s t a r c h . toluene used as a  Soluble starch was used and a drop of  preservative.  Procedure: Before a c t u a l sampling of the gas took place the a s p i r a t o r s were well washed, f i r s t w i t h water and then w i t h starch-iodine  solution.  About  300 ccs. of .1% starch were added t o mixing b o t t l e and iodine  solution  added from the burette u n t i l a l i g h t blue t i n t was abtained.  The mixture  was  then divided between the mixing b o t t l e s i n such a way that the solution  was not  i n contact with the a i r .  In order t o accomplish t h i s , the bent  tube of the test b o t t l e was inserted into the rubber t u b i n g at the end of the bent tube of the mixing b o t t l e and the l a t t e r inverted.  By r e l e a s i n g  the plnchcock on the long tube of the mixing b o t t l e and the short tube of the t e s t b o t t l e the mixture could be made to f l o w from the mixing bottle to the t e s t i n g b o t t l e u n t i l i t reached the mark i n d i c a t i n g 150 ccs. These b o t t l e s were shaken thoroughly.  Th© s o l u t i o n from each t e s t i n g  bottle was introduced into one o f the a s p i r a t o r s a l s o i n such a manner as to prevent contact with the a i r .  The glass rod was removed from the  lower rubber stopper of the a s p i r a t o r and the bent tube of one o f the a«pir&toss inserted.  The t e s t i n g b o t t l e was then inverted and the pinchcock  of the long glass tube and that of the tube i n the upper stopper o f the a s p i r a t o r released so that the starch-iodine aspirator.  s o l u t i o n flowed into the  The t e s t - b o t t l e was then removed, the stopper replaced and the  upper pinchcook closed.  B  o t h a s p i r a t o r s were thoroughly shaken and the  s o l u t i o n returned t o the t e s t b o t t l e s i n a manner s i m i l a r to that described f o r passing the s o l u t i o n from the mixing b o t t l e t o the t e s t - b o t t l e . I f the c o l o r of the s o l u t i o n i n the two b o t t l e s was the same the a s p i r a t o r s were judged ready f o r use.  I f the colour wa? not the same the s o l u t i o n s  were mixed and the procedure repeated u n t i l the same colour was abtained f o r both.  When t h i s c o n d i t i o n had been reached the s o l u t i o n s could be  poured out of the a s p i r a t o r s and sampling could be begun. Both stoppers were removed from the a s p i r a t o r s .  One was placed i n  the cabinet i n which fumigation was to be c a r r i e d on and the other was exposed t o the normal gas mixture of the a i r .  Care was taken i n both  cases to o b t a i n free c i r c u l a t i o n o f the a i r . While the a s p i r a t o r s were being exposed, f r e s h starch i o d i n e s o l u t i o n s were prepared and poured i n t o the t e s t i n g b o t t l e s as p r e v i o u s l y described.  When the sulphur dioxide  had been w e l l mixed with the a i r i n the chamber both a s p i r a t o r s were corked and the one removed from the fumigation iodine solutions were introduced shaken thoroughly.  cabinet.  The s t a r c h -  i n t o the a s p i r a t o r s and these were  The s o l u t i o n s were then poured back i n t o the t e s t i n g  b o t t l e s and compared.  I t was found that the s o l u t i o n from the a s p i r a t o r  exposed to sulphur dioxide was much l i g h t e r i n colour than t h a t from th© other.  This colour difference was due to the a c t i o n of the sulphur  dioxide i n the gas of the cabinet.  Iodine was then t i t r a t e d i n t o the  l i g h t e r s o l u t i o n very c a r e f u l l y u n t i l t h i s s o l u t i o n was brought up to the same coluur as that from the a s p i r a t o r exposed only to the a i r . Since l c . c of fl/500 Iodine s o l u t i o n i s equal to .0224 c.cs. of S0j>  at 0°G and 760 mm.  pressure i t was possible to c a l c u l a t e the proportion  of SOg i n the 8 l i t r e sample under e x i s t i n g c o n d i t i o n s . By t h i s means i t w as determined that w i t h the apparatus being used the a c t u a l conc e n t r a t i o n of sulphur d i o x i d e being abtained was approximately 98% of that expected. 1  This method i s adapted from that used by the Selby Smelter  Commission. Method of Fumigation: The potted trees ware placed i n the cabinet.  A small casserole  containing the mixture of a l c o h o l and carbon b i s u l p h i d e was placed on a shelf i n the cabinet and the door nearly closed l e a v i n g only enough room to i n s e r t a l i g h t e d match to i g n i t e  the a l c o h o l . The cabinet door was  shut t i g h t l y and the e l e c t r i c fan set i n motion. run f o r about ten minutes and then stopped.  The f a n was allowed to  The trees were allowed to  remain i n the chamber f o r a f u r t h e r t h i r t y - f i v e minutes.  I f several  fumigations were to be c a r r i e d out i n quids: succession a t least f i f t e e n minutes of thorough a i r i n g was allowed between fumigations and the trees were removed from the chamber a f t e r the f i n a l fumigation f o r the day completed.  was  I f only one fumigation was being performed the trees were re-  moved a f t e r f o r t y - f i v e minutes fumigations b.  general r e s u l t s  S u p e r f i c i a l symptoms of i n j u r y . In a l l cases the f i r s t sign of i n j u r y i s a general greying of the foliage.  1.  The next stage i s a general browning of the leaves* u s u a l l y  Holmes, J . A., F r a n k l i n , E. C , and Gould, A. Be port o f the Selby SmeIter Commission, Dept. of the Interiors, Bureau of Mines. Government P r i n t i n g O f f i c e , Washington, U. S. A., 1915. pages,*-  s t a r t i n g from the t i p of the needle hut somstimss i n i r r e g u l a r patches over the surface.  Young leaves wither at the t i p s and droop very q u i c k l y .  In some trees such as the Douglas F i r the needles f a l l very r e a d i l y even a f t e r a l i t t l e exposure to sulphur d i o x i d e funas.  The brovaiing i s f o l l -  owed by a bleaching a c t i o n which causes the needles t o become a l i g h t tan shade.  Yihen t h i s stage i s reached there i s u s u a l l y no recovery of  the tree even i f i t i s removed from the fumigation chamber. Buds seem to be very r e s i s t a n t , e v e n s l i g h t l y open buds of the Douglas F i r and Pine showed growth a f t e r the plant had been exposed to fourteen doses of sulphur dioxide i n a concentration of 1 part of SOg i n 10,000, and had appeared completely dead.  I t was noticeable i n the case of the Douglas  F i r that a few of the young leaves were p a r t i a l l y exposed and of these the t i p s were withered while the base continued normal development about f o u r weeks a f t e r the apparantly dead p l a n t had been removed from the cabinet. S p e c i f i c rate of i n j u r y .  '  One of the f i r s t things noticed about the f i v e species used was that sulphur dioxide fumes d i d not e f f e c t a l l of them at the same r a t e . (Tables 1 to 5 ) .  In each fumigation s e r i e s the f i r s t species to show  marked e f f e c t was the Douglas F i r which showed very d i s t i n c t graying of the leaves a f t e r only one or two periods of exposure to sulphur d i o x i d e . (Tables 2 and 3 ) .  In t h i s species a l s o leaves were l o s t or f e l l at a  touch a f t e r two fumigations i n Series 2 (Table 2)and S e r i e s 3 (Table 3) and a f t e r three periods i n Series 4 (Table 4 ) .  The l a s t to show i n j u r y  was the cedar which e x h i b i t s only greying when the Douglas F i r shows l o s s of leaves.  (Tables 1 and 3). A f t e r i n j u r i o u s e f f e c t s commence to become  evident there i s a very r a p i d browning and w i t h e r i n g of the leaves i n the Cedar (Table 4).  The Pine shows browning at the t i p s of the leaves  e a r l i e r than does the cedar, but the progress o f t h i s c o n d i t i o n i s very much slower i n the Pine than i n the cedar (Tables 3 and 4).  The pine  needles while brown at the t i p s and showing greying of the r e s t of the needles remains i n t h i s c o n d i t i o n f o r some time before t o t a l browning occurs.  Hemlock and spruce are intermediate between the Douglas F i r and the  Pine and Sedar.  Hemlock shows i n j u r y a l i t t l e sooner than does the Sprvce  (Tables 2, 5 and 4).  The order i n which i n j u r y i s evinced i s as f o l l o w s ,  Douglas f i r , hemlock, spruce, pine and cedar, while the cedar u s u a l l y shows t o t a l browning sooner than does the pine. (c)  Factors involved i n determining the rate of i n j u r y . It w i l l be n o t i c e d from the accompanying t a b l e s that not only do  the species vary i n t h e i r rate of i n j u r y by sulphur d i o x i d e , but that the time of the f i r s t c l e a r i n d i c a t i o n of i n j u r y to any species v a r i e s i n d i f f e r e n t fumigation s e r i e s .  In S e r i e s 1, i n j u r y i s evident a f t e r four  periods while i n Series 3 one fumigation produces the same r e s u l t .  The  f a c t o r s which are involved i n t h i s d i f f e r i n g rate of i n j u r y w i l l be considered i n the next few  paragraphs.  The f i r s t f a c t o r to be discussed w i l l be the time f a c t o r .  It w i l l  be n o t i c e d that i n those cases which do not show noticeable i n j u r y u n t i l a f t e r the f o u r t h fumigation the fumigation periods were close together, i n f a c t were given i n groups of several i n one day.  Table 1 shows a  condition where four fumigations were given i n one day and Tables 3 and 4 where only one fumigation was given i n one day.  In each case obser-  vations were taken on the morning f o l l o w i n g the fumigation*  Examination  - 14 -  of the tables shows that the degree of i n j u r y i s very s i m i l a r i n both cases.  Injury while not evident a t once develops quite markedly w i t h i n  twenty f o u r hours and i t s degree i s not apparantly increased i n proport i o n by the a d d i t i o n of three or f o u r fumigation periods during the day. Temperature and humidity have been found to have a very decided e f f e c t upon the rate of i n j u r y i n p l a n t s exposed t o sulphur dioxide fumes. 1 Holmes, F r a n k l i n and Gould found that sulphur dioxide fumes were less 2 i n j u r i o u s i n a c o n d i t i o n of low humidity and Zimmerman and Crocker found p l a n t s more r e s i s t a n t i n dry a i r .  Under the c o n d i t i o n s of the  experiment the temperature and humidity v a r i e d considerably from day to day but s i m i l a r conditions obtained i n each fumigation s e r i e s . humidity ranged from 50% to 75%, averaging about 60%.  The  In a l l but the  l a s t two s e r i e s (Series 4 and 5) the temperature range was from 48°0 to o o 80 C w i t h an average temperature of 60 C. Series 4 and 5 were run during a period of e x c e p t i o n a l l y f i n e spring weather so that the temperature o o range i s somewhat higher i n t h i s case being from 52 C to 32 C and averaging  o 65 0.  The humidity, however, remained p r a c t i c a l l y the same as f o r the  previous experiments*  With this wide range of humidity i t was impossible  to draw any conclusions as to the e f f e c t of t h i s f a c t or upon the rate or degree of i n j u r y r e s u l t i n g from fumigation. Temperature appears to have a very great e f f e c t upon the rate of injury.  One experiment (unrecorded because incomplete) was commenced i n  August 1933 and i t was found that a f t e r one exposure to one part of sulphur dioxide i n ten thousand the Douglas f i r l o s t nearly a l l i t s leaves while hemlock, spruce and pine showed almost complete browning and a considerable p o r t i o n of the cedar had turned brown. 1.  Qp. c i t .  2.  Qp.  cit.  The temperature  Date  2 no. Cone.  Time  20.4.33  i  1:10,000  11.15am  tt  2  •i  12.30am  II  3  tt  4  H  0  K  6  It  »i  21.4.33  a(J  1  23.4.33  1.40am D . F . a l l leaves grey to brown dropping  2.45am 'H and C g r e y i n g and brocn P&S s l i g h t " greying. 9.45am  1.45am x/.jr.cteciaea Drowning M. browning.P&S 2.50am &6 browning at t i p s increased S unregular brovmine and bleach i r e . 9»15am  tt :  8  It  9  II  10.20am D.P.appears dead. H. many leaves en11.20ara t i r e l y brown, and S.browning and mottl&ng.C&P d i s t i n c e browning. 10.00am r. nearly dead S. n e a r l y dead. 11.00am G. h a l f dead.  tt  10  Notes.  26.10.33 11  tt  L2  tt  28.10.33 13  tt  10.00am  14  tt  11.00am A l l trees appear dead'.  : lM  i  Abbreviations used throughout t a b l e s H'.. -  Hemlock  S  -  Spruce  DP  -  Douglas F i r  C  -  Cedar  P  -  Pine  Table  2.  R e c o r d o f F u m i g a t i o n S e r i e s No. P.  Date  Ho.  30.10,33  1 :•'  30.10.33  SO OoSc.  Time  Notes  1 20,000  2.00pm  2  •t  3.00pm  D.F. distinct greying leaves f a l l at touch.  31.10.33  3  tt  2.30pm  D.F. leaves browning f a l l at slightest j a r r i n g . S greying ends of branches drooped.  31,10.33  4  tt  3.30pm  1.11.33  5  ii  9.45am'  1.11.33  6  it  10.45am  6.11.33  7  it  1.15pm  7.11.33  8  it  9.25am  G. H & P some yellowing  D. F. many needles f a l l e n H. yellowing. C.P.and S. some yellowing and browning. H - nearly a l l leaves brown c - browning. DP- most leaves f a l l e n P&S browning.  gable  3  Record of Fumigation Series Fo 3. Date  22.1.34  23.1.34  2  27.1.34 •. 3 ;•  Cone.  Time  1 10,000  5pm  ti  4.30pn  it  5.15pm  •.. Notes. D.F.distinct greying. P.S.H. Some greying. 3. s l i g h t greying.  gable  D.F.very grey, leaves drop r e a d i l y , H. greying d i s t i n c t P & S greying more evident 0 greying D.F. most leaves f a l l e n . H & S very brown and grey C & P browning a t ends of leaves C more than P.  4.  Record o f Fumigation Series No. 4. Date  No.  28.4.34 : I  29.4.34  2  2.4.34  3  3.4.34  4  Cone.  1 10,000 1 10,000 1 »o,ooo  Time  Notes  4.30pm D.F.greying n o t i c e a b l y Others s l i g h t l y g r e y i n g C - l i t t l e greying D.Fvery grey 5.30pm H greying P &S some greying C l i t t l e greying 5 pm  DF leaves f a l l r e a d i l y H&S bromiing S P & C browning.  5.15pm  D F leaves f a l l i n g H & S brown. C very brown P n e a r l y a l l brown.  Table 5 Record of Fumigation Series No 5„ Bate 12.4.34  No.  .  ,  15.4.34  2  14.4.34  : 3  15.4.34  Gone.  Time  1 10,000  4.55pm  t i p s of leaves greying  4.00pm  Tips o f leaves browning  it  5.30pm  Leaves brown except a t base  it  4.20pm  Most leaves brown some green at base.  «  4  Notes  0 at t h i s time ranged as h i g h as 96 0 i n the shade and t h i s together w i t h the humid atmosphere o f the greenhouse produced a c o n d i t i o n i n © M e n very r a p i d i n j u r y was obtained. Another important f a c t o r i n determining the rate of i n j u r y i s the co d i t i o n of the p l a n t a t the time of fumigation. In those cases where the trees used had been i n the greenhouse some time and were i n the r a p i d l y growing c o n d i t i o n i n j u r y was r a p i d (Tables 2, 3 and 4 ) .  Much l e s s  r a p i d e f f e c t s r e s u l t e d when t r e e s were allowed to remain i n the greenhouse only long enough to become used t o the change i n atmospheric c o n d i t i o n (Table 5 ) .  Series 4 and 5 are p a r t i c u l a r l y i n t e r e s t i n g since  the temperature and humidity conditions were almost the same f o r both. I n s e r i e s 4. however  9  the trees used had been i n the greenhouse f o r some  time and were i n the r a p i d l y growing condition while i n Series 5 the Douglas F i r used had been i n the greenhouse f o r only a few days.  In  s e r i e s 4 the Douglas f i r showed loss of leaves a f t e r the t h i r d fumig-  a t i o n while i n Series 5 the leaves did not f a l l even a f t e r the f o u r t h 1 period.  I f as F. W. O l i v e r  suggests, i n j u r y occurs through the  en-  trance of sulphur dioxide into the l e a f by the stomata, the growing c o n d i t i o n of the p l a n t may  a f f e c t the rate at which i t may  through the p h y s i o l o g i c a l c o n d i t i o n of the stomata. 2 Haselhoff and Lindau as quoted by Frazer by the whole l e a f surface.  be i n j u r e d  On the other hand,  state that the gas i s absorbed  In t h i s case the c o n d i t i o n of the stomata  would have l i t t l e e f f e c t upon the rate of i n j u r y .  I t i s possible that  when the trees are i n a r a p i d l y growing' c o n d i t i o n , the chemical oontents of the leaves may  be such that the sulphur dioxide on e n t e r i n g the  leaves  may most r e a d i l y reach w i t h and destroy some e s s e n t i a l constituent of the cell.  Whatever the cause, i t seems c l e a r that trees are more e a s i l y i n -  jured i n the r a p i d l y growing condition than they are when p a r t i a l l y or wholly dormant. 2.  H i s t o l o g i c a l studies.  Very l i t t l e l i t e r a t u r e on t h i s phase of the subject was a v a i l a b l e . In most i n v e s t i g a t i o n s a n a l y s i s rather than h i s t o l o g y has been staessed i n the study of t h i s problem and o f those accounts which do deal with 3 the h i s t o l o g y only one or two were obtainable.  Frazer  quotes i n t r a n -  s l a t i o n from Hoselhoff and Lindon's," In the i n t e r i o r of the c e l l p l a s molysis i s induced; the grains of c h l o r o p h y l l are destroyed, and  finally  form w i t h the plasma and other materials a brown amorphous mass. At the 1. 3.  ikp. c i t 2. Op. c i t . Frazer, P. Search f o r causes of Injury to Vegetation i n an Urban V i l l a near a large I n d u s t r i a l Establishment. Transactions of the American I n s t i t u t e of Mining Engineers. V o l . 38. Published by the I n s t i t u t e , New York 1907, page 500.  S&.ES time i n most cases, e s p e c i a l l y i f i n j u r y nas been gradual, tannin separates out as can be demonstrated by brown o r black nodules i n the I  cells."  I n "Urban Fogs"  Oliver  gives two main stages i n the e f f e c t of  the gas on leaves, f i r s t the d e s t r u c t i o n o f c h l o r o p h y l l and second the formation of a d e f i n i t e l a y e r of separation I n the p e t i o l e . of these e f f e c t s may  Either  occur separately o r they may occur i n the same l e a f .  Other w r i t e r s mention plasraolysis as c h a r a c t e r i s t i c of c e l l s i n j u r e d by sulphur d i o x i d e . In  the present study p a r t i c u l a r a t t e n t i o n was p a i d to the s t a r c h  grains and to various p o i n t s which arose i n connect ion w i t h the general d i s o r g a n i z a t i o n of the c e l l s , a.  M a t e r i a l and Methods. A p r e l i m i n a r y experiment was run i n which samples were taken a f t e r  the f i f t h , n i n t h , eleventh and t h i r t e e n t h fumigations.  In s e c t i o n i n g  these i t was found that even as soon as the f i f t h fumigations e s p e c i a l l y i n Douglas f i r , spruce and hemlocks, there were such great changes i n the c e l l contents that l i t t l e s t r u c t u r e could be made out s a t i s f a c t o r i l y * I t was then considered necessary to take samples a f t e r each fumigation and t h i s was done.  Samples were taken i n each case on the morning f o l l -  owing the fumigation. The k i l l i n g and f i x i n g s o l u t i o n used was B. C. F r i a t i v e having the f o l l o w i n g formula. Water ( d i s t i l l e d ) , Methol a l c o h o l 95% Formalin 4-0% Glacial acetic acid  1.  Op. O i t .  100c.cs. 500 c c s . 25 c c s . 25 c c s .  The specimens were dehydrated i n metho1 a l c o h o l and f i n a l l y embedded from S y l o l .  Sections v/e re cut on a r o t a r y microtome.  The m a t e r i a l  was  found to be somewhat b r i t t l e and e a s i l y t o r n so that most of the sect i o n s had to be cut a t between 8 and 15 u\  Gum  arable and  potassium  dichroroate were used as f i x a t i v e s . Two  types of s t a i n i n g of the sections were used.  F i r s t safranin  i n 50% methol a l c o h o l w i t h l i g h t green i n clove o i l as a  counterstain  and then aqueous safranin w i t h a l c o h o l i c gention v i o l e t (50% methol a l c o h o l was used) as a counter s t a i n . the more e f f e c t i v e . In fumigation  The  l a t t e r method was  found to be  The sections were mounted i n Canada balsam.  Series 5, f r e s h material was taken f o r free-hand sec-  t i o n s and these were stained d i r e c t l y w i t h  Iodine.  Study of the l e a f s e c t i o n s : Normal Specimens. In order to describe the e f f e c t s produced by Sulphur diosd^O i t w i l l be necessary f i r s t to out l i n e b r i e f l y the normal c o n d i t i o n of the leave s o In the normal Douglas F i r l e a f the palisade c e l l s are t i g h t l y packed and the spongy mesophyll c e l l s are rather l o o s e l y packed l e a v i n g large sub-stomatal c a v i t i e s . The starch grains are numerous and large and  are  placed p e r i p h e r a l l y around a large c e n t r a l vacuole.  31a)  The epidermis i s one The  (Plates (I) and  l a y e r of c e l l s t h i c k with a heavy smooth c u t i c l e .  stomata are i n shallow, p i t s . In the hemlock the palisade l a y e r i s s l i g h t l y l e s s compact than i n  the Douglas f i r .  The  spongy mesophyll c e l l s are large and l o o s e l y packed  leaving b i g and rather i l l - d e f i n e d sub-stomatal c a v i t i e s . The  starch  -  22  -  p l a s t i d s are large and are so c l o s e l y packed i n a p e r i p h e r a l l a y e r round the c e n t r a l vacuole that they appear hexagonal i n o u t l i n e . (Plates (11) a n d ( X l l ) a . )  ?he surface of the heavy c u t i c l e i s f i n e l y serrated.  The epidermis i s one c e l l i n thickness except at the l e a f margins where a second layer of schlerenchyma c e l l s may  be present.  The stomata are i n  shallow p i b s . The p a l i s a d e l a y e r i n the pine i s very compact while the mesaphyll c e l l s are somewhat l o o s e l y packed.  The sub-stomatal c a v i t i e s are small  and quite d e f i n i t e l y o u t l i n e d by large spongy mesaphyll c e l l s .  The  starch  g r a i n s are found i n a compact p e r i p h e r a l l a y e r g i v i n g a hexagonal appearance to the i n d i v i d u a l g r a i n s . epidermis.  Two  layers o f c e l l s are present i n the  The outer layer c o n s i s t s of small c e l l s w i t h very heavy w a l l s ,  and very s n a i l lumen.  In the inner l a y e r the c e l l s are large and do nfct  possess a very t h i c k w a l l . At the angles o f the leaves there i s occasiona l l y a t h i r d l a y e r of c e l l s s i m i l a r to those i n the second l a y e r . c u t i c l e i s t h i c k and smooth. heavy overhanging c u t i c l e .  The  The deep stomata1 p i t s are protected by  the  (Plates 1? and XI7a.)  No very d e f i n i t e palisade l a y e r i s discernable i n the spruce the ent i r e mesaphyll region being f i l l e d w i t h a f a i r l y compact mass of rather irregular c e l l s .  The starch grains are massed i r r e g u l a r l y i n the c e l l s  (Plates 111 and X l l l a . ) i n strong contrast with the d e f i n i t e p e r i p h i r a l arrangement found i n the Douglas f i r , hemlock and pine.  A large number of  r e s i n c e l l s are present and show dark brown when s t a i n e d . ermis i s d e f i n i t e l y two layered.  The upper epid-  The outer c e l l s are large and t h i c k  walled and the inner c e l l s are sclerenchyma w i t h heavy walls and lumen.  small  D i r e c t l y below the vascular bundle the lower epidermis has  two  -  23-  layers of c e l l s s i m i l a r to those i n the upper epidermis.  Between t h i s  region and the margin of the l e a f the lower epidermis i s only one l a y e r of c e l l s thiclc.  The c u t i c l e i s heavy and smooth.  Stomata are situated  i n rather deep p i t s but have not the overhanging p r o t e c t i v e c u t i c l e found i n the p i n e . The palisade c e l l s i n the cedar are shorter and wider than those i n the other species studied.  They form a compact l a y e r .  The  spongy  mes-  a p h y l l c e l l s are rather l o o s e l y paclsBd but the sub-stomatal c a v i t i e s are small.  The starch grains are arranged p e r i p h e r a l l y . A large number of  h e a v i l y s t a i n i n g r e s i n c e l l s are present.  The upper epidirmis c o n s i s t s of  two l a y e r s of heavy w a l l e d c e l l s , the inner of which are d e f i n i t e l y schlerenchyma c e l l s with very heavy w a l l s and small lumen.  The  lower  epidirmal c e l l s vary i n s i z e and are nonvex on the outer surface g i v i n g , i n s e c t i o n , a scalloped appearance to the lower edge of the l e a f . c u t i c l e i s very thiclc on both upper and lower epidermis.  The  The  scalloped  appearance o f the lower epidermis i s accentuated by the f a c t that the c u t i c l e i n t h i s region i s frequently thickened a t the convex surface of the c e l l so that a protruberance of the c u t i c l e r e s u l t s (Plates Y and  XVa).  Stomata are s i t u a t e d only on the lower surface which i s c l o s e l y a p p l i e d to the stem and they are found i n deep p i t s . I t i s evident i n a l l these c o n i f e r s that the starch grains are contained w i t h i n the c h l o r a p l a s t s , the l a t t e r showing as a l i g h t e r unstained r i n g around the former. Fumigated Specimens: I n examining sections of leaves from t r e e s which have been subjected to fumigations there are two main features which are very evident.  The  - 24 » f i r s t of these features i s the decreased size of the starch g r a i n s .  Table  6 shows the r e s u l t s of a s e r i e s of measurements made from m a t e r i a l which had been k i l l e d and f i x e d i n B. G. f i x a t i v e embedded and stained with s a f r a n i n and gentian v i o l e t .  Measurements i n Table 7 are taken from  Douglas f i r m a t e r i a l which was sectioned by hand i n the f r e s h c o n d i t i o n and stained w i t h i o d i n e . A l l measuremants were taken w i t h the a i d of a camera l u c i d a .  In each case the f i g u r e s r e f e r to diameter i n microns.  The f i v e species w i l l be discussed i n d i v i d u a l y . In Douglas f i r most of the starch grains are large but there i s a great deal of v a r i a t i o n i n s i z e , the smallest being 2.5 H i n diameter and the l a r g e s t 11.0  U,.  Although only subjected to two fumigations the r a p i d  decrease i n the s i z e of the s t a r c h grains i s very n o t i c e a b l e being a r e duction from 6.21 U to 2.04 U..  In t h i s species the amount of the de-  crease i s almost the same during the f i r s t and the second periods.  The  size of the smallest grains appears to remain s t a t i o n a r y during the  two  fumigations but t h i s i s probably only apparent.  The average s i z e of tho  smallest grains i n the normal l e a f i s 2.5 H a n d the average of the smallest g r a i n s a f t e r the f i r s t fumigation i s 1.3 U, a decrease of 1.2 U, i n diameter.  I f a corresponding decrease i n s i z e occured between the f i r s t  and second fumigation tho se starch g r a i n s which a f t e r the f i r s t fumigati oi measured 1.3 IX, i n diameter would be reduced to about. 1 IX. or destroyed altogether.  In Table 7 the reduotion i n the size of the starch grains i s  l e s s marked than i n Table 6. to 3.oi  u;.  In t h i s case the reduction i s from 4.91  U.  6.  Table  Diameters of s t a r c h grains of Douglas F i r , Spruce, Pine, Hemlock and Cedar from needles of trees exposed t o SO . M a t e r i a l k i l l e d and f i x e d i n B. C. f i x a t i v e . Fumig Average diameter at i o n of Starch grains Ho.  Tree Douglas F i r  . : •  Spruce Hemlock  0 1 • 2 ' 3  . 0 • • • • 1 ': 2 3 0 ' 1 -2 3 :  Pine  0 1 a . s  Oedar  .  0 1 2  ! i  i • -S t  Range i n diameter of §tarGli'-graiu.s.»co  6.21 IX 4.01 U 2.04 JU  2.5 XI - l i n 1.3 a - 9.6 IT 1.3 IX 4.5 n  5.03 3.29 2.34 3.02 6.50 4.01 3.98 3.67  2.6 IX 1.31 1.3 IX 1.3 7X 3.9 n 2.6 II 1.9 jDL 1.3 U.  U, T\  R  tt. Uv a.  D<  4.13 Ii, 2.71 DL 2.29 H 2.21 II  1.9 1.3 1.3 1.3  4.80 a 3.00 DL 2.79 JX 2.05 I I  2.6 H 1.9 D, 1.9 II .65U  9.6 II - " . 5.8 I I 3.2 IX - 6.5 H - io.3 a - 6.5 TI - 6.5 U. - 5.8 TX  H JX B, II -  7.8 4.5 3.9 3.9  -  13.6 4.5 5.8 3.2  -  U, IX 0. U IT II 11 II  Table 7. Diameter of stardh grains o f Douglas F i r from needles of trees exposed to S O . M a t e r i a l sectioned f r e s h and stained w i t h Iodine. Tree  Douglas F i r  Fumigation Ho. 0 1 2 3 4  Average diameter of Starch Grains 4.91 4.21 3.34 3.02 , 3.01  &  II IX U, IX  Range i n diameter of Starch g r a i n s . 3.2 2.6 1.9 1.9 1.3  xx Ik Xk -  IX, -  7.0 5.8 4.5 5.2 5.2  11 Ik IX  II AX  - 26 The amount of the change which takes place a t each fumigation i s the same f o r the firstIftvo fumigations,  The f i r s t period caused a reduction  of .7 u and the second o f ,87 TJ. This r e s u l t compairs w i t h that shown i n Table 6 except that i n the l a t t e r case the degree o f reduction i s greater.  As has been p r e v i o u s l y stated that the Douglas f i r used i n  fumigation Series 5 was not i n j u r e d nearly as speedily as were those used the previous experiments. As t h i s i s the case, reduction i n the s i z e of the s t a r c h grains would not be expected t o be as r a p i d as I t was In the previous cases. The hemlock shows reduction from 6.5 U to 3,67 II and reduction i n range from 3.9 H- 10.3 11 to 1.5 1^ - 5.8 U.  The average reduction i s  about one h a l f the normal s i s e and the reduction i n the smallest grains i s two t h i r d s of the normal while the largest are reduced by four f i f t h s . In t h i s case the amount o f reduction i s not nearly equal for each fumiga t i o n as i t was i n the Douglas f i r . The f i r s t period r e s u l t e d i n the reduction of 2.94  i n the diameter o f the grains* the second caused  only .03 I\ reduction, an.amount which i s p r a c t i c a l l y n e g l i g i b l e , while the t h i r d caused a loss of .31 II diameter. Reduction from 5.03 IJ^to 2.34 U i s found i n the spruce.  Here as  i n hemlock the greatest decrease occurs i n the f i r s t fumigation where reduction i s 1.74 Q- the smallest grains are reduced to one h a l f and the largest g r a i n s to one t h i r d of t h e i r normal s i z e .  There i s an  apparent increase i n the size o f the starch grains i n the l a s t fumigation.  2his appearance may be due to the toltal d e s t r u c t i o n of many of  the smaller starch g r a i n s . The pine s t a r c h g r a i n s show decrease i n s i z e from 4.13 L\_to 2.33 II,  -  27 -  and a reduction i n range from 1.9 IX.- 7.8 U, to 1.3  3.9U.  This  species resembles the hemlock and the spruce i n that the greatest r e duction i n diameter o f the grains takes place i n the f i r s t  fumigation.  The l o s s i n t h i s case i s 1.41 D\ i n the f i r s t p s r i o d and .42 IX i n the second while i n the t h i r d i t i s .08 U.  I t i s noticeable that the smal-  l e s t starch g r a i n s i n the pine are considerably smaller than those found i n the other species.  A f t e r the second fumigation no s i g n i f i c a n t r e -  duction appears t o take p l a c e . The cedar shows the greatest v a r i a t i o n i n the s i z e o f the p l a s t i d s . The range i n t h i s species i s from 2.6 II to 13.6 II and the average diameter i s 4.811. The small s i z e of the average g r a i n s as compared w i t h the maximum s i z e shows that the large grains must be small i n numbers. As i n a l l the species, but the Douglas f i r , the greatest reduction i n s i z e i s i n the f i r s t period where the decrease i s 1.8 U.  The f o l l o w i n g period  shows a reduction of only .11 and the t h i r d of .64 U.  The range of var-  i a t i o n i s reduced from 2.6 l{ - 13.6 1^ to .65 L\to 3.2 U, the reduction of the l a r g e r grains i s p a r t i c u l a r l y n o t i c e a b l e .  Unlike the pine and  hemlock, the cedar shows d e f i n i t e decrease i n the s i z e o f the g r a i n s i n the t h i r d fumigation. In comparing the r e s u l t s obtained, as sho\wi i n Table 6, I t w i l l be seen that the degree and r a t e o f the decrease i n the s i z e of the starch g r a i n s i n each species corresponds almost exactly w i t h the order i n which the species show i n j u r y .  The greatest t e t a l reduction i s shown i n  the Douglas f i r where the grains are reduced by about two t h i r d s o f t h e i r normal s i z e a f t e r two fumigations,  i f the other species are also com-  pared a f t e r the second fumigation i t i s seen that Spruce, hemlock and pine  are reduced by approximately one h a l f o f t h e i r normal s i z e while cedar i s reduced by three f i f t h s .  These r e s u l t s suggest that the cedar should  show response to fumigations before the hemlocks spruce and pine, but these species seem to have reached almost the l i m i t of reduction at the second period while the cedar shows d e f i n i t e continued decrease i n the third period. The  second noticeable change i n the c e l l s a f t e r exposure to sulphur  dioxide i s the general d i s o r g a n i s a t i o n of the c e l l contents, and i n some cases of the c u t i c l e . In Douglas f i r fthere i s a noticeable c o l l a p s e of the spongy mesa phy11 and pain sade c e l l s a f t e r one o r two fumigations (plates (VI) and [XI). The c e l l w a l l s become i r r e g u l a r and i n some cases c o l l a p s e d . The reduct i o n i n the s i z e o f the s t a r c h grains has already been, mentioned (Table 6).  Decrease i n s i z e i s accompanied by change i n the shape o f the g r a i n s  which become more l e n t i c u l a r i n shape or even i r r e g u l a r i n o u t l i n e .  The  hexagonal shape due to the compact arrangement of the p l a s t i d s , as mentioned i n the d i s c u s s i o n of the normal h i s t o l o g y of the l e a f , i s l o s t and the p l a s t i d s become widely separated in the c e l l s .  In many c e l l s  the nucleus i s the only d e f i n i t e structure v i s i b l e a f t e r two fumigations. The c e l l contents have become changed to a mass of f i n e l y granular mate r i a l which s t a i n s r e a d i l y w i t h a l c o h o l i c gentian v i o l e t (Plate (XI). Ho e f f e c t i s v i s i l l e i n the epidermis and the c u t i c l e . Plate ( 11) shows the general collapse o f the mesaphyll c e l l s i n the hemlock l e a f a f t e r one fumigation.  The e f f e c t of the c l o s e packing of  the starch p l a s t i d s as seen i n the hesagonal shape produced i s not enti r e l y l o s t although the spacing of the grains i s d i s t i n c t l y wider than i n  the normal c o n d i t i o n .  (Plates (11) 711 and XL1).  The reduction i n the  p l a s t i d s i s not acconrpanied by such narked change i n shape as i s evinced by the Douglas f i r , and only a few of the g r a i n s show i r r e g u l a r o u t l i n e s , i n the hemlock a very d e f i n i t e e f f e c t i s produced upon the c u t i c l e which becomes loosened from the uppsr epidermis p a r t i c u l a r l y , and i s e a s i l y t o r n away i n s e c t i o n i n g (Plate 711). The pine shows rapid d i s o r g a n i s a t i o n of the l e a f t i s sue.  Plasmolysis  i s very evident e s p e c i a l l y i n sorts of the c e l l s , and ths c e l l w a l l s are frequently c o l l a p s e d . The hexagonal appearance of the starch grains i s e n t i r e l y l o s t a f t e r one fumigation.  The g r a i n s become very much smaller  d i s t i n c t l y l e n t i c u l a r and show a dark s t a i n i n g c e n t r a l p o r t i o n surrounded by a comparatively wide l i g h t e r area.  The l i g h t area i s very d e f i n i t e l y  defined i n some cases and i n others i t i s d i f f i c u l t to determine i t s limits.  The nucleus does not show any e f f e c t but the remaining c e l l  con-  tents form an amorphous moss i n contrast w i t h the granular contents of the c e l l s of the Douglas f i r . already described.  Further fumigations only increase the e f f e c t s  There seems to be a continuous breakdown of the c e l l  walls u n t i l no very d e f i n i t e w a l l s can be d i s t i n g u i s h e d a f t e r three fumi g a t i o n s (Plate 217.)  The epidermis and c u t i c l e show no e f f e c t .  D i s o r g a n i s a t i o n of the l e a f t i s s u e i s l e s s evident i n the spruce than i n the preceding species.  The p l a s t i d s are reduced i n s i z e and appear  scattered throughout the c e l l rather than massed together as i n the n o r mal specimens, or i n some cases they appear pushed to the periphera where they form an i r r e g u l a r l a y e r (Plate XL1I).  A few c e l l s show the contents  as a granular mass s i m i l a r to that found i n the Douglas f i r . A few very i n d i s t i n c t p l a s t i d s may he found i n these granular c e l l s .  In s t i l l  other  cases the s t a r c h grains appear to have p a r t i a l l y d i s i n t e g r a t e d i n such a way as to form threads or a network of p l a s t i d m a t e r i a l which s t a i n s red w i t h s a f r a n i n (Plate X I I I ) .  A f t e r two fumigations there i s evidence  of break down of the c e l l w a l l s resembling the c o n d i t i o n found i n Douglas f i r and pine. The cedar does not show much e f f e c t a f t e r the f i r s t fumigation.  After  three p s r i o d s , however, there i s a very d e f i n i t e break down of the l e a f tissue.  The c e l l walls are c o l l a p s e d .  i n s i z e show no p e c u l i a r i t y o f shape.  The p l a s t i d s while being reduced Many o f the c e l l s show d i f i n i t e  plasmalysis but no i n j u r y i s shown by the epidermis and c u t i c l e . The stomata i n almost a l l cases were found closed i n the fumigated specimens suggesting to c o l l a p s e .  that exposure to sulphur dioxide causes these c e l l s  This c o n d i t i o n might be expected i n view of the general  d i s o r g a n i s a t i o n of the other chloraphast containing t i s s u e o f the l e a f . Lack of m a t e r i a l made i t impossible to f o l l o w t h i s l i n e of i n v e s t i g a t i o n to a s a t i s f a c t o r y conclusion so that i t i s possible only to suggest t h i s as a possible r e s u l t of fumigation. The general c o l l a p s e o f the t i s sue suggests that d e s s i c a t i o n i s one o f the e f f e c t s produced and t h i s i s substantiated &y the dry weight determinations made and which w i l l be recorded l a t e r .  The d i s o r g a n i s a t i o n of  the t i s s u e a l s o suggests that some chemical r e a c t i o n has taken place so that the no una 1 contents of the c e l l s are e i t h e r a l t e r e d or p a r t i a l l y destroyed.  In some species, p a r t i c u l a r l y i n the spruce and cedar, there a r e  a number of c e l l s f i l l e d w i t h a dark amorphous mass which i s probably a mucilaginous degeneration product.  - 31 3.  Physiological  stadias  The l i n e of study persued i n the section o f the work was by the h i s t o l o g i c a l studies already c a r r i e d out.  suggested  I t has already been  shown that there i s a great r e d u c t i o n i n the s i z e of the s t a r c h grains a f t e r exposure to sulphure dioxide and this discovery suggested an i n v e s t i g a t i o n o f the e f f e c t of the gas upon the food storage of the l e a f . P a r t i c u l a r s t r e s s was l a i d upon the sugar and s t a r c h r e l a t i o n s i n t h i s regard.  No l i t e r a t u r e wa-& found upon t h i s subject so that a l i n e of  a t t a c k was worked out based upon the s o l u b i l i t y of a v a i l a b l e sugar i n the l e a f and the i n s o l u b i l i t y of the s t a r c h , (a)  Pood Storage M a t e r i a l and Methods.  The m a t e r i a l used f o r fumigation was  taken on the morning a f t e r the  fumigation had been made. The f r e s h leaves were used and e x a c t l y .5 grams taken.  The leaves were cut up f i n e l y and ground i n a mortar w i t h  quartz sand.  To t h i s m a t e r i a l was added 25 c c s . of d i s t i l l e d water and  the whole allowed to stand f o r an hour w i t h occasional shaking.  The  liquid  was then f i l t e r e d o f f and t e s t e d f o r sugar. A ten cubic centimeter sample of the f i l t e r e d liquidwas b o i l e d w i t h 50 c c s . o f a s o l u t i o n of copper sulphate i n potassium carbonate and potassium bicarbonate f o r ten minutes and codled thoroughly.  The p r e c i p -  i t a t e of cuprous oxide was f i l t e r e d out by s u c t i o n through an asbestos mat i n a gooch c r u c i b l e and washed v/ith d i s t i l l e d water.  30 c c s . of hot d i s -  t i l l e d water were poured upon the asbestos mat containing the p r e c i p i t a t e and the whole thoroughly heated.  10 c c s . o f hot f e r r i c s u l f a t e s o l u t i o n  i n s u l p h u r i c a c i d was added to the mixture to d i s o l v e the p r e c i p i t a t e . The s o l u t i o n was then t i t r a t e d with a s o l u t i o n o f potassium permanganate i n which 1 c c . of the permanganate s o l u t i o n was equivalent to 11.7 mg. of copper.  The method described i s a m o d i f i c a t i o n of the Wood-©st copper 1  carbonate method as described by Cole . The s o l u t i o n s used are a s follows Copper carbonate s o l u t i o n potassium carbonate 250 gms. potassium bicarbonate 100 gms. Dissolve i n 600 c.cs. of warm d i s t i l l e d water. C r y s t a l l i n e cdpper sulphate (pure) 23.5 gms. Dissolve i n 200 c.cs. of d i s t i l l e d water. Add the copper sulphate s o l u t i o n to the potassium carbonate and bicarbonate s o l u t i o n slowly mixing the two thoroughly. Make the volume up t o 1 l i t r e and f i l t e r . F e r r i c Sulphate (acid) s o l u t i o n . Add slowly 250 c.cs. of pure concentrated sulphuric acid to 750 c.cs. heat.  of d i s t i l l e d water.  Add f e r r i c sulphate to the s o l u t i o n and  Repeat t h i s u n t i l the presence of a small amount of p r e c i p -  i t a t e shows that the s o l u t i o n i s saturated. Potassium permanganate s o l u t i o n . Dissolve 6 gms. of potassium permanganate i n 1100 c.cs. of cold d i s t i l l e d water and standardize the s o l u t i o n w i t h ammonium o r a l a t e .  Cole, S. V/. P r a c t i c a l P h y s i o l o g i c a l Chemistry. Seventh E d i t i o n , W. H e f f e r and Sons, L t d . , Cambridge, England. 1926, pages 176 to 179.  - 33 -. In determining the starch content the method used was modified from that given i n the United States Department of A g r i c u l t u r e , Bureau of Ch1 emistry B u l l e t i n 107 . Ihe m a t e r i a l from which the s o l u t i o n to he t e s t ed f o r sugar was f i l t e r e d was used f o r the s t a r c h determination.  This  residue was p l a c e d i n an Eslenmeyer f l a s k w i t h 100 c.cs. of 10^ HCL s o l u t i o n and heated on a water both f o r three hours. The l i q u i d remaining' was measured and 10 c.cs. taken f o r a n a l y s i s .  In t h i s sample the a c i d  was almost n e u t r a l i z e d w i t h sodium hydroxide and the sugar content determined as described i n the previous paragraph. Results i  In a l l the r e s u l t s tabulated below the f i g u r e s used r e f e r to m i l l i g r a m s o f copper reduced. Leaves from fumigation Series 3, 4, and 5 were taken f o r a n a l y s i s . M a t e r i a l was taken on the morning a f t e r fumigation i n each case. The r e s u l t s abtained from series 3 are shown i n Tables 8 and S and g r a p h i c a l l y i n Figures 1 to 5. Table 8. Sugar content of .5 gms. of leaves taken from specimens used i n fumigation i n each case. Fumigation numbers Species  0  1  Pine  24.86  43.87  Hemlock  23.4  45.34  Spruce  23.4  43.87  Oedar  29.25  48.26  2 14.62  3 21.94 17.55  24.86  14.62 29.25  4.38 leaves f a l l e n 76.05 Douejlas F i r 34.85 A s s o c i a t i o n of o f f i c i a l A g r i c u l t u r a l Chemists, O f f i c i a l and P r o v i s i o n a l Methods of A n a l y s i s , U. S. Dept. o f A g r i c u l t u r e . Bureau o f Chemistry B u l l e t i n 107 (revised). Government P r i n t i n g O f f i c e , washington,U.S.5. 1912, page 53, 8 (a)  - 34 -  Table 9. Starch content from the same material as used, i n table 8< Fumigation number Species ...Pine. •>  0  1  2  3  ; 122.85  99.91  145.08  150.67  90.09  147.42  187.20  153.97  Spruce  131.62  51.48  87.75  159.47  Cedar  109.69  131.62  —  Douglas F i r  Xe3X ©62  131.62  119.06  Hemlock  148.47  leaves  fallen  The f i r s t st±i«king thing about these results i s the fact that each species shows a very marked increase i n the sugar content after the f i r s t fumigation (Figure 5) and that the decrease i s a linos t as sufi&en after the second period as was the rise a f t e r the f i r s t . Another interesting feature Is the apparent corelation between the rate of injury and the increase of the sugar content. In the Douglas f i r which shows the most rapid injury two and a half times the normal sugar was found a f t e r one fumigation while i n the cedar only one and a half times the normal was found i n the same period.  By examining the  rise i n the sugar content as shown i n Table 8 the following order of i n crease w i l l be shown. Douglas f i r shows the greatest followed by hemlock, spruce, pine and cedar i n descending order. This l i s t w i l l be found to be the same as that given on pagelS where the trees are l i s t e d in the order i n which they show injury.  FIGURE  U  Showing the r e l a t i o n of sugar and starch i n Pine leaves during fumigation with 1 part of sulphur dioxide i n 10,000, .5 gm. of leaves from specimen used i n fumigation Series 3 were analysed i n each oase.  - 36 -  FIGURE 2 .  Showing the relation of sugar and starch i n Hemlock leaves during fumigation with 1 part of sulphur dioxide i n 10,000. .5 gm. of leaves from specimen used i n fumigation Series 3 were analysed i n each case.  - 56 -  FIGURE 3.  Showing the r e l a t i o n o f sugar and starch i n Spruce leaves during fumigation w i t h 1 part of sulphur dioxide i n 10,000. .5 gni. of leaves from the specimen used i n fumigation Series 3 were analysed i n each case.  - 37 -  FIGURE 4.  Showing the relation of sugar and starch i n Douglas F i r leaves during fumigation with 1 part of sulphur dioxide i n 10»000. .5 gm. of leaves from the specimen used in fumigation Series 3 were analysed i n each case.  '- 38 -  FIGURE 5, Showing the v a r i a t i o n i n the sugar content of leaves of Douglas F i r , Hemlock, Spruce and Pine, during fumigation w i t h 1 part of s u l phur dioxide i n 10,000. .5 gm. of leaves from specimens used i n fumigation s e r i e s 3 were analysed i n each case.  In comparing the e f f e c t of fumigation on the starch and sugar content of  the leaves i t w i l l he w e l l to discuss each species separately. The sugar and s t a r c h r e l a t i o n s h i p i n the pine i s shown g r a p h i c a l l y  i n Figure 1 and tabulated i n Sables 8 and 9. The f i r s t fumigation caused a d i s t i n c t r i s e i n t h e su§ar content from 24.86 mg. to 45.87 mg. and a corresponding f a l l i n the starch content from 122.85 mg. to 99.91 mg. the  A t the second fumigation the r e s u l t i s reversed and an increase i n s t a r c h from 99.91 mg. to 145.08 mg. i s accompanied by a decrease  i n the sugar from 45.87 mg« to 21.94 mg. while fete s t a r c h remains almost the  same during the t h i r d p e r i o d the sugar increases t o 21.94 mg. The  source o f t h i s a d d i t i o n a l sugar i s not c e r t a i n but the apparent d i s appearances o f much o f the c e l l w a l l s as diown i n p l a t e (IX) suggests the  p o s s i b i l i t y that some of the c e l l u l o s e may be hydrolyzed t o form  sugars of some sort and thus serve a s the source o f t h i s increase i n sugar.  Another possible source o f the sugar i s the f a t s stored i n the  c e l l s which may be broken down into sugars.  In t h i s s e r i e s the sugar  and s t a r c h reactions of the p:me are the inverse of each other. During the f i r s t p e r i o d the s t a r c h and sugar both increased i n the hemlock (Figure 2, Tables 8 and 9).  During the second p e r i o d however  a c o n d i t i o n s i m i l a r to that i n the Pine i s found, that i s an increase i n the s t a r c h from 147.42 mg. t o 187.2 mg. i s accompanied by a corresponding decrease i n the 45.34 mg. to 26.32 mg.  During the l a s t period  both sugar and s t a r c h are shown to decrease although the rate of decrease i s lessened f o r the sugar i n the t h i r d p e r i o d i n comparison w i t h the second.  The behaviour o f the sugar i n the hemlock i n the l a s t period  suggests a c o n d i t i o n s i m i l a r to that found i n the pine but very much l e s s  - 40 marked and w i t h p o s s i b i l i t i e s of s i m i l a r sources of the a d d i t i o n a l sugar. In the Spruce (Figure 3 and Tables 8 and 9) the sugar and starch are  very c l e a r l y the inverse o f each other.  As the sugar increases from  23.4 mg. to 45.34 mg. i n the f i r s t period the s t a r c h decreases from 131.52 mg. t o 51.48 mg.  In the second period the reverse occurs and the starch  increases t o 87.75 mg* w h i l e the sugar decreases to 24.86 mg. the  During  t h i r d pariod the s t a r c h shows a v e r y marked increase while the sugar  although showing an a c t u a l decrease shows a s l i g h t r e l a t i v e increase. In Douglas f i r the most marked r i s e and f a l l i n the sugar content takes place (Figure 4 ) . During the r a p i d increase of the sugar from 34.85 mg. to 76.05 mg, shown i n tha f i r s t p e r i o d the starch content r e mains p r a c t i c a l l y the saae. the  The r a p i d f a l l i n the sugar content during  second fumigation i s accompanied by a r e d u c t i o n o f the s t a r c h from  131.62 mg. to 119.06 mg. which i s not at a 11 i n proportion with the sugar reduction. The r e s u l t s o f a n a l y s i s from f u m i g a t i o n Series 4 are tabulated i n Tables 10 and 11 and shown g r a p h i c a l l y i n f i g u r e s 6 to 11. Table 10. Fumigation number Species  0  1  2  3  4  Pine  18.95  45.27  49.7  32.17  48.2  Hemlock  13.10  20.47  24.8  59.73  27.2  Sprues  14.62  17.55  26.32  36.5  26.32  Oedar  16.02  23.4  67.27  33.57  26.32  Douglas F i r  26.32  48.2  33.58  40.95  32.17  Numbers r e f e r to mg. o f copper reduced.  Table 11  —*- -*o  "OOU  Xil  X «  Die  XUa  Fumigation number Species  0  1  2  3  4  Pine  167  171.05  258.45  145.1  182.28  Hemlock  .137.59  226.39  232.48  200  196.56  Spruce  193.8  231.46  205.9  142.9  107.54  Cedar  159.12  164.97  146.01  157.95  181.2  Douglas F i r  136.89 ;  117.93  150.9  164.7  163.8  As lias already been shown i n the d i s c u s s i o n o f Table 4 the specimen s i n fumigation S e r i e s 4 d i d not show such r a p i d i n j u r y as, did those i n Series 3.  I t i s i n t e r e s t i n g to n o t i c e that the period of maximum sugar  content i s a l s o delayed i n a l l except the Douglas f i r .  (Figure I I ) .  Cedar and pine show maximum sugar content a t the second period and heml o c k and spruce a t the t h i r d .  The Douglas f i r , although i t does not show  the greatest sugar increase i n t h i s s e r i e s i s the f i r s t to reach i t s  maximum. In the pine the sugar shows an increase from 18.95 mg, to 45.27 ng. i n the f i r s t period while the starch shows a r e l a t i v e decrease although a s l i g h t a c t u a l increase was n o t i c e d .  Daring the second period the  rate of sugar increase i s very much l e s s than i n the f i r s t while the starch shows a marked increase from 111.05 mg. to 258.45 ag. Both the sugar and s t a r c h decrease i n the t h i r d period and increase i n the fourth, but the r e s u l t s of these periods may not be of much value, e s p e c i a l l y those from the f o u r t h period since the t r e e s were almost dead by t h i s time.  - 42 In the cedar, the a n a l y s i s shows the expected r e s u l t s a f t e r the f i r s t period.  During the second p e r i o d the sugar increases r a p i d l y from  23.4 mg. to 67.27 mg. while the s t a r c h decreases from 164.97 mg. to 145. 01 mg. She. sugar shoWB a decrease from 67.27 mg. to 33.57 mg. i n the t h i r d period and a f u r t h e r decrease to 26.32 mg. i n the f o u r t h .  The  starch on the other hand shows a s l i g h t increase i n the f o u r t h p e r i o d from 157.95 mg. to 191.2 mg.  (Figure 7 ) .  In the Hemloclc (Figure 8 and Tables 10 and 11) the sugar and starch content i s increased during the f i r s t two periods although the starch shows a g r e a t e r inorease during the f i r s t period than does the sugar and a smaller increase than the sugar i n the second period.  Daring the  t h i r d and f o u r t h periods the usual r e s u l t s a r e abtained. The increased sugar content from 24.8 mg. t o 59.73 mg. i n the t h i r d period i s accompanied by a decrease i n the s t a r c h content from 232.48 mg. to 200 mg. while i n the f o u r t h period the opposite occurs when the s t a r c h shows a r e l a t i v e increase while the sugar shows a decrease to 27.75 mg. (Figure 8 ) . The sugar and starch both show an increase i n the f i r s t period i n the  spruce. During the sedond and t h i r d periods the sugar shows a c t u a l  increase from 17.55 mg. to 25.32 mg. and i n the t h i r d period to 36.5. The s t a r c h shows a corresponding decrease from 231.46 mg. to 205.9 mg. i n the second fumigation and from 205.9 mg. to 142.9 mg. i n the t h i r d . During the f o u r t h p e r i o d the sugar shows a decrease from 36.5 mg. to 26,32 mg. while the r a t e of starch decrease i s s l i g h t l y l e s s .  These r e -  s u l t s suggest the inverse r e l a t i o n s between starch and sugar found i n the other species.  During the f i r s t p e r i o d the s t a r c h and sugar both show an increase i n the Douglas f i r .  (Figure 6 and Tables 10 and 11).  During the second  period decrease i n sugar content from 48.2 mg. to 38.58 mg. panied by increased s t a r c h from 117.93 mg. to 150.9 mg.  i s accom-  In the t h i r d  fumigation the s t a r c h shows a r e l a t i v e decrease while the sugar increases to 40.95 mg.  I t i s doubtful i f the r e s u l t s of the f o u r t h period are  valuable as s t a t e d i n the case of the pine sine© by t h i s time many of the leaves had f a l l e n or were i n a c o n d i t i o n to f a l l at the s l i g h t e s t touch or j a r r i n g .  The r e s u l t s show the inverse r e l a t i o n between the  starch and sugar to soma extent but i t i s not as d e f i n i t e i n the Douglas f i r as i n some of the other species. Results of a n a l y s i s of specimens from fumigation Series 5. The only species used i n t h i s s e r i e s was the Douglas f i r .  The  tree was i n the greenhouse long enough to become a c c l i m a t i s e d but not i n a r a p i d l y growing condition as were those i n Series 3.  was  Injury  e f f e c t s i n t h i s case were much slower in showing themselves since the leaves were not l o s t even a f t e r f o u r fumigations whereas i n Series 3 and 4 the Douglas f i r showed loss-fOf' leaves i n the second and t h i r d periods r e s p e c t i v e l y . The r e s u l t s are shorn i n Table 12 and Figure 12. Table 12. Sugar and starch content of .5 gms. of leaves taken from Douglas f i r used i n fumigation Series 5. M a t e r i a l taken on the morning a f t e r the fumigation i n each case.  Stegar Starch  ;  o  204.75  1 • 35.1 245.7  Furoiga t i o n number 2 3 4 43.87 35.1 49.72 215.38 246.6 285.01  - 44 numbers r e f e r to mgs. of copper reduced. The climax i n sugar increase i s reached a t the t h i r d fumigation i n t h i s case which shews delayed e f f e c t s since i n Series 5 and 4 Douglas f i r showed the climax at the f i r s t period of fumigation.  In the second  period the sugar increases from 35.1 mg. to 43.8 mg. and the s t a r c h decreases from 245.7 mg. to 215.38 mg. while during the t h i r d period both increase.  During the l a s t fumigation the r e s u l t s are the opposite of  those found i n the second, the sugar decreases from 43.72 mg. to 35.1 mg. and the s t a r c h r i s e s from 246.6 mg. to 285.01 mg. e f f e c t o f the s t a r c h and sugar are observable  The usual inversed  i n t h i s s e r i e s as i n those  previously described although there i s a s l i g h t i r r e g u l a r i t y introduced i n the t h i r d p e r i o d where both s t a r c h and sugar show increase. Discussion of R e s u l t s . It i s notticeable i n each case recorded that there i s a very r a p i d and marked increase i n the sugar content of the leaves a f t e r one o r two fumigations w i t h one part of sulphur dioxide i n ten thousand.  This con-  d i t i o n suggests that one o f the primary e f f e c t s o f the sulphur dioxide gas i s the a c t i v a t i o n of the enzyme re sponsible f o r the production o f sugar.  The period over which the a c t i v a t i o n i s e f f e c t i v e v a r i e s with the  condition under which fumigation i s c a r r i e d out. The r e s u l t s of fumiga t i o n Series 3 (Figure 5) show that the peak of a c t i v a t i on i s reached a f t e r one fumigation i n a l l species.  In Series 4 (Figure 11) the maximum  was reached a t the f i r s t fumigation i n the case of Douglas f i r , the second i n the case of cedar and pine and the t h i r d i n the case o f Spruce and hemlock.  I t i s c l e a r that i n t h i s s e r i e s some f a c t o r causes a delay i n  - 45  ~  FIGURE 6*  Showing the r e l a t i o n o f sugar and s t a r c h i n Pine leaves during fumigation w i t h 1 part Sulphur dioxide i n 10,000. .5 gm. of leaves from specimen used i n fumigation Series 4 were analysed i n each case.  - 46 ~  FIGURE 7.  Showing the r e l a t i o n of sugar and starch i n Cedar leaves during fumigation w i t h 1 part of Sulphur dioxide i n 10„000. .5 gm. of leaves from specimen used i n fumigation Series 4 were analysed i n each ca.se o  FIGURE 8.  Showing the r e l a t i o n o f sugar and s t a r c h i n Hemlock leaves during fumigation with 1 part of sulphur dioxide i n 10,000. .5 gm. of leaves from specimen used i n fumigation Series 4 were analysed i n each case.  FIGURE 9.  Showing the r e l a t i o n o f sugar and s t a r c h i n Spruce leaves during fumigation w i t h 1 part of sulphur dioxide i n 1^,000. .5 gm. of l e a v e s from specimen used i n f u m i g a t i o n S e r i e s 4 were a n a l y s e d i n e a c h case.  » 49 -  FIGURE 10. Showing the r e l a t i o n of sugar and s t a r c h i n Douglas F i r leaves during fumigation w i t h 1 part of sulphur d i o x i d e i n 10,000. x5 gm. of leaves" from specimen used i n fumigation Series 4 were analysed i n each case.  FIGURE 11.  Slowing the v a r i a t i o n i n the sugar content of leaves of Douglas F i r , Hemlock, Spruce, Pine, and Cedar during fumigation with 1 part of sulphur d i o x i d e i n 10,000. z5 gm. of leaves from specimsns used in fumigation Series 4 were analysed i n each case.  FIGURE 12.  Showing the r e l a t i o n of sugar and s t a r c h i n Douglas F i r leaves during fumigation w i t h 1 part o f sulphur d i o x i d e i n 10,003. x5 gm. of leaves from specimens used i n fumigation s e r i e s 5 were analysed i n each case.  - 52 the usual a c t i o n .  Once the maximum i s reached there i s a sharp cirop  i n the sugar content which suggests the p a r t i a l or complete destruction of the enzyra©. I t has "been suggested p r e v i o u s l y that the delayed a c t i o n of the sulphur dioxide was due to the p h y s i o l o g i c a l condition o f the •fersae-at-• at the time of fumigation.  In Series 3 the trees were i n a r a p i d l y grow-  ing c o n d i t i o n owing to having been i n the greenhouse f o r a  considerable  time before being subjected to treatment, while in Series 4 they had been i n the greenhouse only a short time.  Tables 9, 10, 11 and 12, show  that i n f vunigation Series 4 the starch content of the leaves was  notic-  eably greater than i n those from Series 3 i n a l l cases except the Douglas 1 fir.  According to Haas and H a l l  t h i s c o n d i t i o n i s c o r r e l a t e d w i t h the  amount amount of diastase in the leaves for i n t h e i r book on the chemist r y of plant products they make the f o l l o w i n g statement regarding  diastase?  nThe amount present i n any p a r t i c u l a r organ v a r i e s with the conditions o b t a i n i n g ; thus when the temperature and other f a c t o r s are most favorable f o r growth and f o r the germination of starchy seeds, diastase i s much more abundant than when growth and germination are s l u g g i s h .  A l s o , the amount  of diastase i s always greater in s t a r c h leaves than i n sugar leaves, and the same holds f o r i s o l a t e d leaves containing much s t a r c h , as compared w i t h shaded leaves containing l i t t l e or no starch."  I f this c o r r e l a t i o n  between starch content and diastase content holds good i t would suggest an explana t i o n o f the delayed i n j u r y observed i n fumigation s e r i e s 4.  These leaves  showed a higher starch content and should, therefore, also have a higher diastase content than those of Series 3. It seems f e a s i b l e that the 1. Haas, P. and H»11,T.G., An Introduction to the Chemistry of Plant Produ c t s , Longmans, Green & Co., London 1913,pages 358 and 359.  d e s t r u c t i o n of the smaller amount of diastase i n the leaves of the trees used i n Series 3 would be more r a p i d than the d e s t r u c t i o n of the larger amount i n the leaves of the t r e e s used i n Series 4. be born out by the a c t i o n of Douglas f i r .  This theory seems to  The starch Gontent o f t h i s  species i n both s e r i e s 3 and 4 i s almost the same, which suggests equal diastase content and should be accompanied by s i m i l a r reaetions to s u l phur d i o x i d e .  Examination o f Figures 4 and 10 r e v e a l that while the  other species show delayed r e a c t i o n i n Series 4, the Douglas f i r a t t a i n s maximum sugar a f t e r the f i r s t fumigation i n both Series 3 and 4. In Series 5 however, Douglas f i r shows a much l a r g e r proportion o f starch to sugar than i n the previous cases and the maximum sugar content i c not reached u n t i l the t h i r d fumigation p e r i o d .  Another point o f inter-,  est i s shown by the r e s u l t s obtained with spruce, hemlock,cedar and pine. The l a s t two species named show a smaller starch increase than do the f i r s t two. Figure 11 shows that i n the pine and cedar the maximum sugar i s only delayed u n t i l the second fumigation while i n the Spruce and heml o c k which show the greatest starch increase the maximum sugar i s delayed u n t i l the t h i r d p e r i o d . (b)  Dessication Studies. M a t e r i a l and Methods.  M a t e r i a l f o r these studies consisted of leaves taken from a Douglas f i r on the morning a f t e r fumigation i n each case. c a r r i e d out.  In the f i r s t experiment approximately  Two experiments were .5 gms. of leaves  were f i n e l y cut up and accurately weighed i n a o r u c i b l e . On the second experiment branches which had shown the same rate of growth the -previous  - 54 fear were s e l e c t e d and one hundred loaves frora each taken f o r weighing. In each case was p l a c e d i n an oven which was kept a t 100°C f o r four days and was then reweighed i n order to determine the amount of water l o s t from the leaves during d r y i n g .  Results. The data obtained from these two experiments upon the d e s s i c a t i o n e f f e c t s o f sulphur dioxide are given tolow i n Tables 15 and 14. Table  15.  Results of experiment to i n v e s t i g a t e the d e s s i c a t l o n e f f e c t s of S,0g. Experiment 1. Fumigation Ho.  V/t. of green l v s .  Loss of wt. on drying $ l o s s i n wt.  0  •3550 gm.  .2144 gm.  58.74$  1  .5779 gm.  .3347 gm.  57.39$  2  .4754 gm.  .2265 gm.  47.51$  3'  .4486 gm.  .1886 gm.  42.04$  4  .3600 gm.  .1213 gm.  33.07$  Table 14. Results of experiment to i n v e s t i g a t e the d e s s i c a t i o n e f f e c t s of S, 0^ Experiment 2. Fumigation Ho.  Wt. of green l v s .  Loss of Wt. on drying % l o s s i n V/t.  ©  .6212 gm.  .3790 gm.  61.01$  •1  .5959 gm.  .3246 gm.  54.47$  2  .6061 gm.  .2790 gm.  46.03$  3  .4716 gm.  .1917 gm.  40.64$  - 55 -  The r e s u l t s f o r the two experiments, a s given above, agrees i n showing a steady decrease i n the water content of the leaves a s fumigation proceeds. A c c o r d i n g t o t h e "mass law" l o s s o f water i n t h e l e a v e s s h o u l d r e -  s u l t i n r e d u c t i o n o f sugar and increase i n starch but t h i s does not  a c t u a l l y occur u n t i l a f t e r the sugar has reached i t s maximum increase. These r e s u l t s suggest t h a t t h e r e i s some f a c t o r i n v o l v e d w h i c h i n t e r -  f e r e s w i t h the usual w o r k i n g o f the "mass law".  In the d i s c u s s i o n of  the r e s u l t s obtained from work on sugar and s t a r c h a n a l y s i s i t i s suggested  t h a t t h e i n t e r f e r i n g f a c t o r i s t h e a c t i v a t i o n o f a n enzyme or  enzymes. When t h i s a c t i v a t i o n ceases due to the d e s t r u c t i o n of the enzyme or of the a c t i v a t o r the "mass law" asserts i t s e l f and there i s an immediate i n c r e a s e i n the s t a r c h w i t h c o r r e s p o n d i n g decrease i n t h a sugar i n t h e l e a v e s ( F i g u r e s 1 - 4  and 6 ~ 10').  S U M M A R Y 1.  The species used f o r these experiments show d e f i n i t e  injury  when exposed to an atmosphere containing sulphur dioxide gas. 2.  The order i n which the species show i n j u r y i s as f o l l o w s . Pseudotsuga t a x i f o l i a (Lambert) B r i t t o n . (Douglas F i r $ . Tsuga heterophylla, Sarg., (Western Hemlock) Picea s i t c h e n s i s , Carr., (Setka Spruce ) Pinus monticola, Dougl., (Western Y/hite Pine) Thuja p l i c a t a , Donn., (Western Had Oedar)  3.  Injury shows e x t e r n a l l y by g r e y i n g of the f o l i a g e followed  by bleaching and browning and i n sons cases by l o s s of leaves. 4. E n v i r o n m e n t a l f a c t o r s such a s temperature a f f e c t the r a t e of i n j u r y . 5. the  I f one fumigation i s given d a i l y the r e s u l t a n t i n j u r y appears  same as i f s e v e r a l fumigations a r e given i n one day. 6.  Injury shows i n t e r n a l l y by the reduction i n the s i z e of the  starch grains followed by the g e n e r a l degeneration o f the c e l l contents and the collapse of the c e l l w a l l s . 7.  P h y s i o l o g i c a l i n j u r y i s shown by the great i n c r e a s e i n the  sugar content of the leaves followed by a very sudden decrease i n sugar a f t e r the maximum has been reached. 8.  The rate of i n j u r y i s r e l a t e d to the p h y s i o l o g i c a l condition of  the trees, trees w i t h a high sugar content i n comparison w i t h the starch show more r a p i d i n j u r y than t h o s e i n w h i c h the r e v e r s e i s the c a s e .  9.  There i s a steady decrease i n the water content of the leaves  dtTring f u m i g a t i o n .  - 57 -  SOME SUGGESTED PROBLEMS FOR FURTHER STUDY ARI3IKG OUT OF THE ABOVE EXPERIMENTS. 1.  While the starch g r a i n s continue to decrease i s s i z e (Tables 6 and  7) the s t a r c h t e s t shows an a c t u a l increase i n the starch content of the l e a v e s (Tables 9 a n d 11).  Again the sudden r i s e i n sugar content i s out  of proportion to the decrease i n the s i z e of the s t a r c h g r a i n s .  The  source of t h i s increase i n s t a r c h and sugar has not been investigated and might prove a valuable l i n e of research. 2.  Another i n t e r e s t i n g problem a r i s e s out of the c o r r e l a t i o n of the  p h y s i o l o g i c a l c o n d i t i o n o f the tree and the r a t e a t which i t shows i n j u r y . Involved i n t h i s question i s the suggested connection between the prop o r t i o n of s t a r c h i n the leaves, and consequently of the amount of d i a s t ase present, w i t h the rate of iasjury. 3.  The method by which the sulphur dioxide enters the l e a f i s another  l i n e of i n v e s t i g a t i o n suggested. 4.  The i n v e s t i g a t i o n of the a c t i v a t i o n and d e s t r u c t i o n of the enzymes  i n the l e a f would be another i n t e r e s t i n g study. 5.  The general loss o f colour i n the leaves suggests that one of the  f i r s t r e s u l t s of fumigation i s the p a r t i a l or complete d e s t r u c t i o n of the c h l o r o p h y l l . Spectroscopical a n a l y s i s of c h l o r o p h y l l from fumigated leaves would be a f r u i t f u l subject f o r research.  EXPLANATION OP PLATES  PLATE 2.  Cross s e c t i o n of a normal Douglas F i r l e a f .  PLATE I I .  Cross s e c t i o n of a normal Hemlock l e a f .  PLATE I I I .  Cross s e c t i o n of a normal Spruce l e a f . (This material was very b r i t t l e and e a s i l y t o r n . The s e c t i o n shows t h e general arrangement o f the t i s s u e and the massed c o n d i t i o n of the c h l o r o p l a s t s . )  PLATE 17.  Cross s e c t i o n of a normal Pine l e a f .  PLATE 7.  Cross section of a normal Cedar l e a f .  PLATE 71.  Cross section of a Douglas F i r l e a f a f t e r two fumigations w i t h one part of sulphur dioxide i n 10,000.  PLATE 7 1 I I .  Cross s e c t i o n of a l e a f of Hemlock a f t e r two fumigations w i t h 1 part of sulphur dioxide i n 10,000.  PLATE 71II.  Cross s e c t i o n of a leaf of Spruce a f t e r three fumigations w i t h 1 p a r t of sulphur dioxide i n 10,000.  PLATE I X .  Cross s e c t i o n of a l e a f of Pine a f t e r two fumigations with 1 p a r t of sulphur dioxide i n 10,000.  PLATE A .  Cross s e c t i o n of a l e a f of Cedar a f t e r two f u m i g a t i o n s w i t h 1 part of sulphur dioxide i n 10,000.  PLATE S I .  Douglas F i r . (a)  (b-c)  Part of a section of a normal leaf showing the arrangement of the s t a r c h grains a n d the nature o f the upper epidermis. Parts of a section of a leaf a f t e r two fumiga t i o n s w i t h 1 part o f sulphur dioxide i n 10,000. (bJ Palisade c e l l s i n which the contents have degenerated to form a granular mass, (c) Palisade and spongy mesophyll c e l l s showing the general d i s o r g a n i z a t i o n of the tissue.=The starch grains a r e r e duced i n s i z e and i n numbers.  EXPLANATION OF PLATES (d) PLATE X I I . (a-b)  Section of a normal sterna. Hemlock. Parts of a s e c t i o n of a normal l e a f . (a) Palisade and spongy mesophyll c e l l s showing the arrangements of the starch g r a i n s . The upper epi.demis i s a l s a shown. (b) Section of a normal stoma.  (c)  Part of a s e c t i o n of a l e a f a f t e r two fumigations with 1 p a r t of sulphur dioxide i n 10,000. Palisade c e l l s and spongy mesophyll c e l l s showing the reduct i o n i n the s i z e and number of the s t a r c h g r a i n s .  PLATE X I I I . Spruce. (d-g) Parts of a section of a l e a f a f t e r two fumigations w i t h 1 part of sulphur dioxide i n 10,000. (d) A mesophyll c e l l i n which the starch grains have p a r t l y d i s i n t e g r a t e d and have formed a network of m a t e r i a l . (e) A mesophyll c e l l i n which the contents have degenerated into a granular mass. (f) A mesophyll c e l l i n which the starch grains are reduced i n number and are s c a t t e r e d throughout the c e l l . PLATE XIV. (a-b)  Pine P a r t s of a s e c t i o n of a normal l e a f . (a)  (b) (o-d)  Part of a s e c t i o n showing the nature of the upper epidermis and the arrangement of the starch grains i n the palisade and sp&ngy mesophyll c e l l s . Section of a normal stoma.  Parts of a s e c t i o n of a l e a f a f t e r two fumigations with 1 part of sulphur d i o x i d e i n 10,000. (c) Spongy mesophyll c e l l i n which the contents have degenerated i n t o a granular mass (d) Palisade and spongy mesophyll c e l l s showing the reduction and nature of the s t a r c h grains a f t e r fumigation.  ESPLAIATOT OF PLATES  PLATE XT".  {a - b)  Cedar.  Parts o f a s e c t i o n of a normal l e a f . (a) Part of a section showing the arrangement of the s t a r c h g r a i n s i n the p a l i s a d e and spongy mesophyll c e l l s . The tws l a y e r e d e p i d e r m i s i s a l s o shown.  (cj  Part o f a s e c t i o n o f a l e a f a f t e r two fumigations w i t h 1 part of sulphur dioxide i n 10,000.  Some o f the c e l l s show the reduction i n the number and s i z e o f the starch grains while i n others the contents have degenerated i n t o a granular mass. M a g n i f i c a t i o n o f P l a t e s I to X - X 210 M a g n i f i c a t i o n of PlatSs XI to XV - X 190.  PLATE I I  PLATE I ?  PLATE V I  PLATE 711I  PLATE X.  PLATE X I I  PLATE XV  BIBLIOGRAPHY A s s o c i a t i o n of O f f i c i a l A g r i c u l t u r a l Chemists  O f f i c i a l and P r o v i s i o n a l Methods of A n a l y s i s . U. S. Dept. of A g r i c u l t u r e Bureau of Chemistry, B u l l . No. 107 (revised!. Government P r i n t i n g O f f i c e Washington, U. S. A. 1912. s  Bakke, A. L.  - The E f f e c t of C i t y smoke on Vegetation. A g r i c u l t u r a l Experiment S t a t i o n , Iowa State College of A g r i c u l t u r e and the Mechanic A r t s . Ames, Iowa. 1913 - The E f f e c t of Smoke and Gases on Vegetation, Contribution of the Botanical Department, Iowa State College, Ames, Iowa.  Cole, S. W.  - P r a c t i c a l P h y s i o l o g i c a l Chemistry, Seventh E d i t i o n , W. H e f f e r and Sons, L t d . , Cambridge, England. 1926.  Crowther, C and RustonA.- The Nature, D i s t r i b u t i o n and E f f e c t s upon Vegetation of Atmospheric i m p u r i t i e s i n and near an i n d u s t r i a l Town. Journal of A g r i c u l t u r a l Science V o l . 4, U n i v e r s i t y Press, Cambridge, England, 1911. Ebaugh, E. W.  Frazer, P.  Gases Vs. S o l i d s * An i n v e s t i g a t i o n of the i n j u r i o u s ingredients of smelter smoke, Journal of the American Chemical Society, V o l . 29. The Chemical P u b l i s h i n g Co. Easton, Pa. 1907. Search f o r causes o f Injury to Vegetation i n an Urban V i l l a near a large I n d u s t r i a l E s t a b l i s h ment . Transactions of the American I n s t i t u t e of Mining Engineers, V o l . 38. Published by the I n s t i t u t e , New York, 1907.  Haas,P. and H i l l , T . G .  An Introduction to plant Chemistry. Green and Go. London, 1913.  Hamberiandt, G.,  P h y s i o l o g i c a l Plant Anatomy, McMillan and Co. Ltd., London, 1914.  Haywood, J . K.,  Injury to Vegetation by Smelter Fumes, Dept. of A g r i c u l t u r e , Bureau of Chemistry, B u l l 89. Government P r i n t i n g O f f i c e , Washington, U. S. A. 1905.  Longmans  Injury to Vegetation and Animal l i f e by smelter wastes, U. S. Dept. of A g r i c u l t u r e , Bureau of Chemistry, B u l l . No. 113 (revised) Government P r i n t i n g O f f i c e , Washington, U. S . A.  Haywood, J . K.  s  Smelter Smoke, Science V o l . 26. M c M i l l a n Co., New Y o r k ,  Heald, P.  G.,  Manual of Plant D i s e a s e s , McGraw - H i l l Book Co. Inc., Hew York, 1926.  Holmes J . , F r a n k l i n E. Gould, A.  McLell'iuid. E. H.  duller  s  W.  and  Report o f the Selby Smelter Commission U. S. Dept. of the I n t e r i o r , Bureau of Mines, B u l l . 98., Gov. P r i n t i n g O f f i c e Washington, U. S. A. 1915.  Injury through Smoke and F l u Dust, U. S. Dept. o f A g r i c u l t u r e Experiment S t a t i o n Record, V o l . 25. Government P r i n t i n g O f f i c e , Washington, 1912. Urban Fogs. Journal of the Royal H o r t i c u l t u r a l Society, V o l . 16. Spottiswold and Co., London. 1893.  Sorauer, P.,  Zimmerman, P. W., Crocker, W.  s  Bibliography of Smoke and Smoke preventions U n i v e r s i t y o f P i t t s b u r g h , P i t t s b u r g h , Pa.,  M. 0. et a l ,  O l i v e r , F.  1907  Manual of Plant Diseases, (Third E d i t i o n ) , Vol. 1, B e r l i n , Translated by F. Dor ranee, 1922. and  Sulphur Dioxide Injury to P l a n t s . Procodings of the American Society f o r Horti c u l t u r a l Science. Published by the Society, Geneva, New York, 1931.  

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