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Nature of ice-sheet injury to forage plants Freyman, Stanislaw 1967

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The U n i v e r s i t y o f B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE '  FINAL ORAL  EXAMINATION  FOR THE DEGREE OF' DOCTOR OF PHILOSOPHY  of  STANISLAW  FREYMAN  B. S c . ( A g r i c . ) U n i v e r s i t y of P r e t o r i a , 1959 M.S.A. U n i v e r s i t y  o f B r i t i s h Columbia, 1963  FRIDAY, MARCH 3, 1967 AT 3:30 P.M. IN ROOM 102, AGRICULTURE BUILDING  COMMITTEE IN CHARGE Chairman: V. J. A. D.  B. N. Moyls  C. B r i n k R. MacKay McLean P. Ormrod External  R. A. Rowles W. B. S c h o f i e l d C. P. S. T a y l o r  Examiner:  Department  Dale Smith  o f Agronomy  University of Wisconsin Madison Research S u p e r v i s o r :  V. C. B r i n k  '  THE  NATURE OF  ICE-SHEET INJURY TO  FORAGE PLANTS  ABSTRACT The n a t u r e of i c e - s h e e t damage to o v e r w i n t e r i n g forage p l a n t s was s t u d i e d i n a c o n t r o l l e d environment a t n o n - i n j u r i o u s f r e e z i n g temperatures. The s o i l atmosphere was analyzed i n a gas-chromatograph and the p l a n t s were assessed f o r i n j u r y by h i s t o l o g i c a l examination and r e c o v e r y r a t e s i n the greenhouse. Under experimental i c e - c o v e r s carbon d i o x i d e accumulated i n the s o i l i n some i n s t a n c e s to as h i g h as 107° while oxygen was d e p l e t e d to l e s s than 47o of the atmosphere P l a n t s r o o t e d i n such s o i l s were k i l l e d a f t e r 7 weeks of i c e - c o v e r . When the s o i l under the i c e - s h e e t was f l u s h e d w i t h carbon d i o x i d e the p l a n t s were k i l l e d a f t e r p e r i o d s as s h o r t as 21 days. In both cases i n j u r y appeared to be p h y s i o l o g i c a l r a t h e r than m e c h a n i c a l . Furthermore, carbon d i o x i d e accumulation r a t h e r than oxygen d e p l e t i o n was r e s p o n s i b l e f o r the i n j u r y s i n c e the p l a n t s were a b l e to withstand p e r i o d s of 3 weeks i n a n i t r o g e n - s a t u r a t e d s o i l . A f r e e z e ~ t h a w - f r e e z e c y c l e , w i t h moderate f r e e z i n g temperatures and a s s o c i a t e d w i t h an i c e - s h e e t , d i d not appear to be damaging to a l f a l f a . Continuous i c e - c o v e r s r e s u l t e d i n a g r e a t e r accumulation of carbon d i o x i d e and c o n s e q u e n t l y more i n j u r y s u f f e r e d by the p l a n t s than where the cover was t e m p o r a r i l y broken by a thaw. H i g h s o i l - m o i s t u r e c o n d i t i o n s which are u s u a l l y a s s o c i a t e d w i t h i c e - s h e e t s d i d not r e s u l t i n an i n c r e a s e d h y d r a t i o n l e v e l i n the t i s s u e and c o n s e q u e n t l y d i d not make the p l a n t s more s u s c e p t i b l e to c o l d i n j u r y . A technique was developed to determine the a b i l i t y of p l a n t s to withstand ice-encasement. S e v e r a l v a r i e t i e s and s p e c i e s that were t e s t e d e x h i b i t e d no c l e a r - c u t c o r r e l a t i o n between r e s i s t a n c e to i c e encasement and frost hardiness.  AWARDS 1963  -  1964-1965  N.O.C.A. S c h o l a r s h i p N a t i o n a l Research C o u n c i l of Canada S t u d e n t s h i p  GRADUATE STUDIES Field  of Study:  Plant  Ecology  Plant Ecology C o n t r o l l e d Environment S t u d i e s Plant Physiology P l a n t Taxonomy & F i e l d Botany Permafrost & D i f f e r e n t Aspects, of Frozen Ground Photogrammetry, P h o t o i n t e r p r e t a ^ t i o n & F i e l d Techniques  V. C. B r i n k D. P. Ormrod D. J . Wort K. Beamish J . R. MacKay A. L. F a r l e y J . Stager  PUBLICATIONS S. Freyman and V. C. B r i n k , Note on an A d d i t i o n to the B i o c h e m i s t r y and G e n e t i c s of Coumarin i n Sweet C l o v e r . Can. J . P i t . S c i . 43: 415-416, 1963. V. C. B r i n k , J . R. MacKay, D. Pearce, S. Freyman, Needle Ice Development i n South Western B r i t i s h Columbia. Can. J . P l a n t S c i . , 1967. (In Press)  THE NATURE OF ICE-SHEET INJURY TO FORAGE PLANTS  ty STAUISLAW FREYMAN. B . S c . j A g r i c . ) , U n i v e r s i t y o f P r e t o r i a , 1959 M.S.A.,. U n i v e r s i t y o f B r i t i s h Columbia, 1963  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF -THE- REQUIREMENTS- FOR"THE DEGREE OF .DOCTOR OF.PHILOSOPHY  i n the D i v i s i o n of P l a n t Science  We accept t h i s t h e s i s as conforming to the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA January,  1967  In p r e s e n t i n g  this  thesis  for an advanced degree at that  tha L i b r a r y s h a l l  study thesis  in p a r t i a l  f u l f i l m e n t of the  the U n i v e r s i t y of B r i t i s h  make i t  I f u r t h e r agree that  freely available  permission  for  requirements  Columbia,  I  agree  r e f e r e n c e and  f o r e x t e n s i v e copying of  this  f o r s c h o l a r l y purposes may be granted by the Head of my  Department or by h i s or p u b l i c a t i o n of without my w r i t t e n  Department of  representatives.  this  thesis  SCtEVcc  Tlvj U n i v e r s i t y o f B r i t i s h Vancouver 8 , Canada  is understood that  for financial  permission.  fUrYl^^  It  Columbia  gain s h a l l  copying  not be allowed  iii  Chairman: P r o f e s s o r Vernon C. Brink  ABSTRACT  The nature of ice-sheet damage to overwintering forage p l a n t s was studied i n a c o n t r o l l e d environment a t non-injurious f r e e z i n g temperatures. The  s o i l atmosphere was analyzed  assessed  i n a gas-chromatograph and the plants were  f o r i n j u r y by h i s t o l o g i c a l examination and recovery r a t e s i n the  greenhouse. Under experimental  ice-covers carbon dioxide accumulated i n the  s o i l i n . some instances to as high as lOfo while oxygen was depleted to l e s s than  of the atmosphere.  7 weeks of i c e - c o v e r .  P l a n t s rooted i n such s o i l s were k i l l e d a f t e r  When the s o i l under the ice-sheet was f l u s h e d with  carbon dioxide the p l a n t s were k i l l e d a f t e r periods as short as 21 days. In both cases i n j u r y appeared to be p h y s i o l o g i c a l r a t h e r than mechanical. Furthermore, carbon dioxide accumulation r a t h e r than oxygen d e p l e t i o n was responsible f o r the i n j u r y since the plants were able to withstand of 3 weeks i n a nitrogen-saturated A freeze-thaw-freeze  periods  soil.  c y c l e j ' with moderate f r e e z i n g temperatures  and associated with an ice-sheet, did^not appear to be damaging to a l f a l f a . Continuous ice-covers r e s u l t e d i n a g r e a t e r accumulation of carbon dioxide and consequently more i n j u r y s u f f e r e d by the p l a n t s than where the cover was temporarily broken by a thaw.  High s o i l - m o i s t u r e c o n d i t i o n s which are  u s u a l l y a s s o c i a t e d with ice-sheets d i d not r e s u l t i n an  increased.hydration  l e v e l i n the t i s s u e and consequently d i d not make the p l a n t s more s u s c e p t i b l e to c o l d i n j u r y . A technique was developed to determine the a b i l i t y of plants to  withstand ice-encasement.  Several v a r i e t i e s and species that were tested  e x h i b i t e d no c l e a r - c u t c o r r e l a t i o n between r e s i s t a n c e to i c e encasement and f r o s t  hardiness.  TABLE OF CONTENTS Section  Page  INTRODUCTION  1  1.  REVIEW OF LITERATURE  3  1.1.  The Growth of Ideas about the Nature of Winter I n j u r y  3  1.2.  A C l a s s i f i c a t i o n of Cold I n j u r i e s  4  1.3.  Opinions on the Cause of Ice-Sheet Damage t o P l a n t s  12  1.4.  Relevant S o i l Atmosphere S t u d i e s  16  2.  3.  MATERIALS AND METHODS  21  2.1.  Boxes w i t h Temperature C o n t r o l and P o t s  21  2.2.  Temperature, P r e s s u r e and M o i s t u r e Measurement  23  2.3.  P l a n t Species and V a r i e t i e s  24  2.4.  S o i l and S o i l Atmosphere Sampling Tubes  25  2.5.  S i m u l a t i o n of Ice-Sheets  26  2.6.  S o i l Atmosphere Sampling and A n a l y s i s  27  2.7.  Injury C r i t e r i a  28  2.8.  Root Measurements  29  EXPERIMENTATION  36  3.1.  Experiment I  36  3.2.  Experiment I I  37  3.3.  Experiment I I I  39  3.4.  Experiment IV  40  3.5.  Experiment V  41  3.6.  Experiment VI  43  3.7.  Experiment V I I  44  vi  Page 4.  OBSERVATIONS AND RESULTS  49  4.1.  Experiment I  49  4.2o  Experiment I I  51  4.3.  Experiment I I I  58  4.4.  Experiment TV  63  4.5.  Experiment V  68  4.6.  Experiment VI  71  4.7.  Experiment VII  72  5.  DISCUSSIONS  74  6.  SUMMARY AND CONCLUSIONS  89  7.  BIBLIOGRAPHY  91  vii  LIST OF TABLES Table No. 4.1.  Page Composition of the s o i l atmosphere w i t h and without an i c e - c o v e r a f t e r 38 days a t -3.0°C.  50  4.II.  Composition o f the s o i l atmosphere a f t e r 50 days a t -3.0°C.  60  4.III.  Composition of the s o i l atmosphere a f t e r 40 days a t -3.0°C. ( i ) Continuous i c e - c o v e r w i t h p l a n t s e n t i r e l y covered and w i t h stubble p r o t r u d i n g through the i c e . ( i i ) Ice-cover i n t e r r u p t e d by a 10 day thaw p e r i o d .  64  The weights o f CO^ l i b e r a t e d when i c e covered s o i l s were f l u s h e d w i t h N^, 0^, and C O ^ - f r e e - a i r .  72  S u r v i v a l o f d i f f e r e n t species and v a r i e t i e s f o l l o w i n g i c e encasement f o r 60 days.  73  6.IV. 7.V.  viii  LIST OF FIGURES  F i g u r e Ko.  Page  2.1  Insulated box w i t h temperature c o n t r o l .  30  2.2  S i n g l e pot. mounted i n box with temperature c o n t r o l .  31  2.3.  Arrangement  2.4  2.5  2.6  2.7  2.8  o f boxes with temperature c o n t r o l on  Dexion frame.  32  P a i n t e d p l a s t i c pot with top sealed, mounted i n CO^ f i l l e d p l e x i g l a s s chamber. Gases i n the pot were withdrawn through a s i l i c o n e septum mounted i n the upper end of the tube p r o t r u d i n g through the l i d .  33  Rotary switch connecting.11 thermocouples mounted i n d i f f e r e n t l o c a t i o n s . Darker cords seen on lower r i g h t connect Bouyucous moisture b l o c k s i n the s o i l beneath the i c e - s h e e t .  33  Arrangement of boxes with temperature c o n t r o l on the Dexion i r o n frame i n the c o l d room. A continuous i c e sheet covering two adjacent boxes can be seen on the surface of the upper boxes. A c y l i n d e r of CO^ was used i n s o i l f l u s h i n g experiment.  34  Manometers were attached to f l u s h i n g tubes to detect any pressure change that may have taken place i n the s o i l under an i c e - s h e e t .  34  Thawing-lid, i n s u l a t e d and equipped with two 60 watt l i g h t bulbs, which provided enough heat to r a i s e the temperature abOT6s8;:,p£ts to 1 5 C . Thermocouple wires lead i n t o the cover f o r temperature measurement. Temperature under the cover was c o n t r o l l e d by a rheostat connected to the l i g h t bulbs. An ice-sheet can be seen i n the lower l e f t corner.  35  Polyethylene l i n e d c r a t e s used i n Experiment V I , to determine the e f f e c t of s o i l moisture on the h y d r a t i o n l e v e l of t i s s u e of overwintering p l a n t s .  42  F l a s k s of cone. H^SO and long white tubes of anhydrous CuSO were used to dry the outflowing gas. COg was then c o l l e c t e d on " A s c a r i t e " packed i n t o small separatory funnels seen above. Small tubes a l s o packed with "Ascarite™ were attached to the o u t l e t end of the separatory funnels to ensure that no atmospheric CO^ would be absorbed.  44  ;  3.1  3.2  ix F i g u r e No. 3.3  3.4  Page H e a v i l y i n s u l a t e d sand box used t o freeze the s o i l i n pots from the surface down. Temperatures a t d i f f e r e n t depths i n the sand were measured w i t h thermocouples mounted a t d i f f e r e n t depths i n the sand. The s o i l o f the 4 potted p l a n t s on the r i g h t i s f r o z e n w h i l e the 4 pots on the l e f t a r e e n t i r e l y surrounded by i c e . A l l 8 pots were s t o r e d a t -3-0 C f o r 2 months. 1  3.5  4.1  4.2  4.3  4.4  4.5  4.6  4.7  46  47  The wax c a r t o n was t o r n o f f immediately a f t e r the pots had been removed from the -3.0 C room, so t h a t water from the m e l t i n g i c e would not f l o o d the p l a n t s . I c e encased pot immediately a f t e r the c a r t o n had been removed can be seen i n the center.  48  Growth o f the 4 p l a n t s a f t e r an i c e - s h e e t cover o f 38 days, was slower than the growth o f the p l a n t s t h a t were not covered by i c e .  50  I c e covered s o i l through which CO^ was f l u s h e d r e s u l t e d i n the death o f the p l a n t s . When 0^ was f l u s h e d through the s o i l , the p l a n t s recovered as v i g o r o u s l y as those which had no I c e - c o v e r .  52  C r o s s - s e c t i o n o f the r e g i o n j u s t below the crown o f an a l f a l f a p l a n t encased i n i c e f o r 62 days. The s o i l was f l u s h e d w i t h 0 . Some mechanical damage t o the phloem and p e r i c y c l e can be seen, (p) p e r i c y c l e , (ph) phloem, ( c ) cambium, (x) xylem ray.  53  C r o s s - s e c t i o n o f the r e g i o n j u s t below the crown o f an a l f a l f a p l a n t kept f o r 62 days a t -3.0 C without an i c e - c o v e r . No mechanical damage i s v i s i b l e .  53  V i t a l s t a i n i n g o f a s e c t i o n taken j u s t below the crown of a p l a n t kept f o r 62 days covered by an i c e - s h e e t . CO was f l u s h e d p e r i o d i c a l l y through the s o i l . The y e l l o w s t a i n i n g t i s s u e i s dead. Ro l i v e , r e d s t a i n i n g c e l l s could be found, (y) y e l l o w s t a i n i n g t i s s u e .  54  V i t a l s t a i n i n g o f a s e c t i o n taken j u s t below the crown of a p l a n t kept f o r 62 days covered by i c e . The s o i l was f l u s h e d p e r i o d i c a l l y w i t h 0^. The deep r e d staining tissue, i s a l i v e . . (r) red s t a i n i n g t i s s u e . V i t a l s t a i n i n g o f a s e c t i o n taken j u s t below the crown of a p l a n t kept f o r 62 days a t -3.0 C without an i c e cover. Most o f the xylem r a y c e l l s a r e a l i v e and s t a i n red.  54  55  X  Figure No. 4.8  4.9  4.10  4.11  4.12  4.13  4.14  4.15  4.16  Page The s o i l i n the 4 pots was covered by an i c e - s h e e t . The s o i l of the 2 pots on the l e f t was aerated by a continuous stream of a i r , while the 2 pots on the r i g h t were p e r i o d i c a l l y f l u s h e d with CCv,.  55  V i t a l s t a i n i n g of a s e c t i o n just, below,,.the crown of a plant kept f o r 21 days covered by an ice-sheet and the s o i l f l u s h e d continuously with a i r .  56  V i t a l s t a i n i n g of a s e c t i o n j u s t below a plant kept f o r 21 days covered by an s o i l f l u s h e d i n t e r m i t t e n t l y w i t h CO^. made a f t e r an 8 day recovery p e r i o d i n  56  the crown of ice-sheet and the Sections were the greenhouse.  An ice-sheet l a s t i n g 7 weeks r e s u l t e d i n the death of the p l a n t s rooted i n both a saturated and i n a s o i l at approx. f i e l d c a p a c i t y . A e r a t i o n of the i c e covered s o i l by l e a v i n g the f l u s h i n g tubes open, prevented t o x i c accumulation of CO^.  61  V i t a l s t a i n i n g o f a s e c t i o n o f a plant kept f o r 50 days a t - 3 . 0 ° C without an ice-sheet cover. S e c t i o n s were made f o l l o w i n g a 10 day recovery p e r i o d . Red s t a i n i n g c e l l s are i n d i c a t i v e of l i v e t i s s u e .  61  V i t a l s t a i n i n g of a s e c t i o n of a plant covered by an ice-sheet f o r 50 days w i t h f l u s h i n g tubes l e f t open to aerate the s o i l beneath the i c e . S e c t i o n s were made f o l l o w i n g a 19 day recovery p e r i o d . Many red s t a i n i n g c e l l s are v i s i b l e , ( r ) red s t a i n i n g tissue.  62  V i t a l s t a i n i n g of a s e c t i o n of a plant covered by an ice-sheet f o r 50 days. The s o i l moisture was at approximately f i e l d c a p a c i t y and the f l u s h i n g tubes were kept c l o s e d . Carbon d i o x i d e accumulated to over lOfo of the s o i l atmosphere. Only a few red s t a i n i n g c e l l s could be detected„ (y) yellow staining tissue.  62  Growth of new shoots at the end of a 10 day p e r i o d under the thawing cover. P r i o r to the thawing period, the p l a n t s on the l e f t were covered by an ice-sheet f o r 20 days, while the ones on the r i g h t were exposed to the same f r e e z i n g temperature, but had no i c e - c o v e r .  65  Due to an i n t e r r u p t i o n i n the i c e - c o v e r by a thaw p e r i o d , CO^ d i d not accumulate to the same extent as i n the pots which were continuously covered by an i c e - s h e e t , and as a r e s u l t , the thaw appears to have been b e n e f i c i a l r a t h e r than more damaging to the p l a n t s . Stubble p r o t r u d i n g through the i c e ,  F i g u r e No. 4.16 (cont'd) 4.17  4.18  4.19  4.20  4.21  4.22  Page apparently d i d not b e n e f i t the p l a n t s to withstand long periods o f i c e - c o v e r .  66  V i t a l s t a i n i n g o f a s e c t i o n taken j u s t below crown o f a plant exposed t o a 20 day f r e e z e , day thaw and a 10 day freeze p e r i o d , without i c e - c o v e r . Sections were made a f t e r 8 days i n the greenhouse.  66  the 10 any recovery  V i t a l s t a i n i n g o f a s e c t i o n o f a plant exposed to a 20 day f r e e z e , 10 day thaw and a 10 day freeze p e r i o d , with an ice-sheet cover during the two freeze periods. Sections were made a f t e r 8 days recovery i n the greenhouse.  67  V i t a l s t a i n i n g of a s e c t i o n taken j u s t below the crown • of a plant encased f o r 40 days i n an ice-sheet, with the stubble protruding through the i c e . Sections were made a f t e r an 8 day recovery p e r i o d .  67  V i t a l s t a i n i n g o f a s e c t i o n taken j u s t below the crown of a plant e n t i r e l y encased i n an ice-sheet with no p a r t s protruding through the i c e .  68  E f f e c t of s o i l moisture on t i s s u e moisture and consequently on the extent of c o l d i n j u r y .  69  Response of s e c t i o n s of p l a n t s stored f o r 48 hours at -10.0°C t o v i t a l s t a i n i n g ; (a.),.(b.) and ( c ) . P r i o r to the f r e e z i n g treatment the plants were kept f o r 12 days a t near f r e e z i n g temperature i n a saturated s o i l ( a . ) , a s o i l a t approximately f i e l d c a p a c i t y ( b . ) , and a dry s o i l ( c ) . Sections o f the p l a n t s exposed t o ' s o i l s a t d i f f e r e n t moisture l e v e l s r e v e a l some l i v e t i s s u e , mostly dead t i s s u e and some mechanical d i s r u p t i o n . . There appears to be no c l e a r c o r r e l a t i o n between the amount o f damage to the p l a n t s and the moisture l e v e l o f the s o i l .  71  xii  ACKNOWLEDGEMENTS  Appreciation Dr. V.C. B r i n k  (Chairman), D i v i s i o n of P l a n t S c i e n c e ,  D i v i s i o n of P l a n t S c i e n c e , and  i s extended t o my graduate committee: Dr. D.P. Ormrod,  Dr. W.B. S c h o f i e l d , Department of Botany  B i o l o g y , Dr. C A . Rowles, Department of S o i l  Science,  Dr. J.R.  Mackay, Department o f Geography, Dr. C.P.S. T a y l o r , Department of P h y s i c s , U n i v e r s i t y of B r i t i s h Columbia, and Mr. A. McLean,  Canada  Department of A g r i c u l t u r e , R e s e a r c h S t a t i o n , Kamloops, B.C.  F i n a n c i a l a i d , g r a t e f u l l y acknowledged, has been provided the  by the N a t i o n a l R e s e a r c h C o u n c i l of Canada through support of  p r o j e c t and through graduate s c h o l a r s h i p f o r two years.  S p e c i a l thanks f o r the use of cold-room f a c i l i t i e s and for  t h e i r kind co-operation  a r e t o be accorded:  Dr. R.E. F i t z p a t r i c k  ( D i r e c t o r ) , and Dr. R. Stace-Smith, Canada Department of A g r i c u l t u r e , Research S t a t i o n , Vancouver, B.C.  T e c h n i c a l a s s i s t a n c e , g r a t e f u l l y acknowledged, has been provided  by Mr. Ilmars D e r i c s , t e c h n i c i a n , Mr. Don Pearce, t e c h n i c i a n ,  and Mr. Dave Armstrong, t e c h n i c i a n , D i v i s i o n of P l a n t U n i v e r s i t y of B r i t i s h Columbia.  Science,  1  INTRODUCTION  The seriousness o f winter damage to crops has been appreciated by man f o r many c e n t u r i e s , but he b e l i e v e d l i t t l e could be done to ameliorate the e f f e c t s of f r e e z i n g temperatures and o f t e n f e l t that severe winters were God's punishment f o r h i s s i n s . Winter damage, d i r e c t l y o r i n d i r e c t l y a t t r i b u t a b l e to low temperature, to crops grown i n north temperate due to pests, diseases and weeds.  l a t i t u d e s may exceed the combined damage F o r an example, o r c h a r d i s t s i n the t r e e -  f r u i t growing area of the i n t e r i o r o f B r i t i s h Columbia,  f i n d that winters  with damaging f r o s t s occur with s u f f i c i e n t frequency, to make winter i n j u r y the main hazard i n f r u i t production.  Winter k i l l i n g i s a l s o one of the most  common causes o f the l o s s o f stands of a l f a l f a throughout growing areas o f North  the major a l f a l f a  America.  Recently there has been an e v e r - i n c r e a s i n g i n t e r e s t i n the nature of winter damage and i n f i n d i n g ways of reducing i t s s e v e r i t y through plant breeding and improved  cultural practices.  Of the c u l t u r a l p r a c t i c e s , ways  and means of modifying the c r i t i c a l s o i l - t o - a i r i n t e r f a c e would be of prime importance.  F o r an example i n Canada, legume-grass mixtures are o f t e n  p r e f e r r e d to pure legume stands from the viewpoint o f winter s u r v i v a l .  By  p r o v i d i n g e x t r a winter cover, the grass helps i n c o l l e c t i n g snow which i n t u r n provides i n s u l a t i o n against extreme c o l d or against a l t e r n a t e f r e e z i n g and  thawing. Winter i n j u r y to herbaceous  p l a n t s may be a t t r i b u t e d to complexes  and i n t e r a c t i o n s of c l i m a t i c f a c t o r s , which i n part i s the reason why, i n  2 s p i t e of extensive  scientific  i n j u r y are u s u a l l y d i f f i c u l t  information about w i n t e r k i l l , the causes of to define p r e c i s e l y .  Of the many f a c t o r s  i n v o l v e d , the one chosen f o r study i s the e f f e c t of an ice-sheet at noni n j u r i o u s temperatures on the gaseous composition  of the s o i l atmosphere,  and t o determine the nature of the damage s u f f e r e d by overwintering  plants  rooted i n the i c e covered s o i l . Although a great deal of work has been done on gas exchange between s o i l and a i r , e a r l i e r s t u d i e s were beset with t e c h n i c a l d i f f i c u l t i e s and, thus, the matter of the composition  of s o i l a i r , e s p e c i a l l y i n winter under  covers such as i c e - s h e e t s , has been l e f t i n an i n d e c i s i v e s t a t e . Recent developments i n techniques,  one o f which i s the use of gas-  chromatography i n the study of s o i l a i r , made a f r e s h approach p o s s i b l e , to r e s o l v e whether or not oxygen d e f i c i e n c y o r carbon dioxide t o x i c i t y or both, developed under i c e - s h e e t s of prolonged d u r a t i o n . Techniques used i n v o l v e d encasing o f herbaceous p l a n t s i n a r t i f i c i a l i c e - s h e e t s , n e c e s s i t a t e d by the rather.sporadic occurrence o f n a t u r a l i c e sheets, sampling o f s o i l gases f o r gas-chromatographic analyses and a s s e s s i n g i n j u r y to the p l a n t s by h i s t o l o g i c a l examination and recovery rates i n the greenhouse.  3  1.  1.1.  REVIEW OP LITERATURE  THE GROWTH OF IDEAS ABOUT THE NATURE OF WINTER INJURY  A r i s t o t l e s a i d , " L i f e cannot e x i s t without i t s n a t u r a l heat."  Low  temperature e f f e c t s on p l a n t s a l s o a t t r a c t e d the a t t e n t i o n o f Theophrastus who s t a t e d t h a t , "The myrtle t r e e i s without heat and t h e r e f o r e f r e e z e s r a p i d l y ; the l a u r e l on the other hand i s r e s i s t a n t as i t r e t a i n s i t s heat." ( l 3 4 ) Damage t o p l a n t s hy low temperatures i s a common phenomenon, and has been a concern o f man s i n c e and even before the e a r l i e s t p r a c t i c e o f a g r i culture.  One o f the e a r l i e s t p r e c i s e d e s c r i p t i o n s o f w i n t e r k i l l i n g o f p l a n t s  was t h a t o f Jacob Bobart ( l 6 ) i n 1683.  Bobart.published  h i s observations i n  the then a u t h o r i t a t i v e , " P h i l o s o p h i c a l T r a n s a c t i o n s , " London, where he wrote t h a t "The extream c o l d and f r o s t happening i n our time, gave occasion o f t a k i n g n o t i c e o f some o f i t s e x t r a o r d i n a r y e f f e c t s T h e  intention of his  d e s c r i p t i o n was "To g i v e some r e l a t i o n o f i t s workings among vegetables, and  4  to i n c i t e others f o r the f u t u r e to the c o n s i d e r a t i o n of the procedure of the c o l d , and how  i t operates upon such bodyes, that thereby we may,  being f o r e -  warned, be i n some measure prepared f o r the b e t t e r p r e s e r v a t i o n and of many t h i n g s i n that nature i n other winters,  defence  the mildest and best whereof,  proves troublesome to many p l a n t s , though i n ' g e n e r a l i t does kindness to e a r t h , and  i t s f u t u r e products."  A few years l a t e r i n 1708/1709 Western  Europe again s u f f e r e d a severe winter which was (4?).  the  described  i n d e t a i l by Deghan  He wrote t h a t , "But among a l l the s u f f e r e r s by the f r o s t , the vegetables  were the most u n i v e r s a l . " times has been recorded T h i s widespread and  The  occurrence of such severe winters i n h i s t o r i c  by Brooks ( 2 5 ) i n h i s book "Climate Through the Ages."  conspicuous winter damage has been most aptly, described  i n a few words by Maximov (87) who  stated t h a t , "Seldom does the  opportunity  occur to observe such pronounced changes i n nature as a f t e r a sudden f i r s t frost.  P l a n t s that were p r e v i o u s l y b u r s t i n g with l i f e now  o f f e r a picture  of complete d e s t r u c t i o n . "  1.2  A CLASSIFICATION OF COLD INJURIES  Exposure to low of ways.  temperature may  Because of i n t e r a c t i o n among the f a c t o r s involved i n winter damage,  t h e i r c l a s s i f i c a t i o n i s d i f f i c u l t , and to determine.  1.2.1.  cause i n j u r y to p l a n t s i n a number  The  Injury Due  the exact cause of i n j u r y o f t e n hard  f o l l o w i n g are ways i n which c o l d may  cause damage to p l a n t s .  to Ice C r y s t a l Formation Within the P l a n t  P l a n t p a r t s or whole p l a n t s may  be k i l l e d  or damaged beyond r e p a i r  5 as a r e s u l t of f r e e z i n g of the t i s s u e s .  I t i s w e l l e s t a b l i s h e d that f r e e z i n g  damage i s caused by the formation of i c e c r y s t a l s w i t h i n the p l a n t s (l31> 88, 80, 81, 89). the  protoplast.  118,  Ice c r y s t a l s may form i n the i n t e r c e l l u l a r spaces and/or i n The former.occurs mainly during a slow drop i n temperature,  while the l a t t e r occurs when the temperature drops r a p i d l y below the f r e e z i n g point.  1.2.1.1.  Slow drop i n temperature.  U s u a l l y when temperatures drop slowly, i c e c r y s t a l s form i n the i n t e r c e l l u l a r spaces which r e s u l t s i n a vapor pressure drop below that of the  p r o t o p l a s t and water d i f f u s e s from the p r o t o p l a s t through the permeable  plasma membrane to the r e g i o n of c r y s t a l l i z a t i o n „(80).  In t h i s way the c e l l  sap concentration increases s t e a d i l y , reducing i t s f r e e z i n g temperature, and ice  does not form i n the p r o t o p l a s t even at very low temperatures (80).  There are two p r i n c i p a l ideas as to how the protoplasm of the c e l l i s i n j u r e d by such dehydration accompanying c e l l u l a r spaces. i n 1933.  i c e c r y s t a l formation i n the i n t e r -  One i s the mechanical theory proposed f i r s t by I l j i n  According to h i s theory withdrawal of water from the c e l l  (58)  subjects  the  protoplasm to p h y s i c a l s t r e s s e s that lead to t e a r i n g and breaking of  the  protoplasm.  The second i s the p r o t e i n p r e c i p i t a t i o n theory proposed  by Sachs ( l i o ) i n 1873.  He suggested that the concentration of the p r o t e i n  i n the protoplasm i n c r e a s e s with the withdrawal of water, to the extent that the p r o t e i n s no longer remain dispersed and begin to coagulate.  This  theory has more r e c e n t l y been r e f i n e d and substantiated through experimental work by L e v i t t et a l . ( 8 2 , 83, 84) and Schmutz et a l . ( i l l ) .  They have  proposed that f r e e z i n g i n j u r y i s due to an u n f o l d i n g and denaturation of the  protoplasmic p r o t e i n s , as water moves to form i c e c r y s t a l s i n the i n t e r c e l l u l a r spaces.  T h i s denaturation of p r o t e i n r e s u l t s from the formation  of i n t e r m o l e c u l a r d i s u l f i d e (S-S) bonds induced by the c l o s e approach of the p r o t e i n molecules during dehydration and consequently death occurs.  1.2.1.2.  Rapid drop i n  temperature.  Rapid f r e e z i n g causes i c e to form w i t h i n the c e l l , both w i t h i n and outside the vacuole.  Ice w i t h i n the protoplasm  probably causes most f r e e z i n g  i n j u r y presumably because c r y s t a l growth d i s r u p t s protoplasmic o r g a n i z a t i o n . (18, 79, 114,  1.2.2.  117.)  " C h i l l i n g " In.iurv  Pjamage from low temperature i s not always due to below f r e e z i n g .  temperatures  P l a n t s of t r o p i c a l and s u b - t r o p i c a l o r i g i n can be i n j u r e d  by " c h i l l i n g " ( l l 6 ) .  T h i s r e s u l t s from s e v e r a l days of exposure to temp-  eratures a l i t t l e above f r e e z i n g (0.5°-10°C).  Thermophilic plants.such as  cucumber, tobacco, corn, cotton, and r i c e are most s u s c e p t i b l e to c h i l l i n g damage.  Corn, f o r an example begins to show i n j u r y at 3.0°C.  The a c t u a l  cause of c h i l l i n g . i n j u r y i s not known, however, L e v i t t ( 8 l ) put forward a number of suggestions: (a)  A higher rate of t r a n s p i r a t i o n than water absorption  (b)  A higher r a t e of r e s p i r a t i o n than photosynthesis or disturbance w i t h i n the former  (c)  A higher r a t e of p r o t e i n breakdown than synthesis  7  1.2.3•  Heaving  1.2,3.1.  Heaving due to i c e lens formation i n the s o i l .  Heaving i n j u r y to p l a n t s i s caused by t h e i r being l i f t e d upward from t h e i r normal p o s i t i o n , causing t h e i r r o o t s to loosen or break.  A f t e r heaving,  i t may be d i f f i c u l t f o r the r o o t s to become f i r m l y e s t a b l i s h e d again and the plant may  d i e from the mechanical damage s u f f e r e d , from d e s i c c a t i o n , or as a  r e s u l t of a pathogen which entered v i a the damaged t i s s u e .  Heaving i s not  caused by expansion of s o i l water f r e e z i n g i n. s i t u i n the s o i l but by f r e e water segregating to form i c e lenses.  interstices,  Thus, an i c e lens obtains  water f o r growth by d r y i n g the s o i l i n i t s v i c i n i t y , thereby c r e a t i n g a s u c t i o n gradient i n the water of the unfrozen s o i l .  F r o s t heaving w i l l only  occur i f s u f f i c i e n t water i s a v a i l a b l e , i f the s o i l temperature  i s below 0°C,  i f the l a t e n t heat of c r y s t a l l i z a t i o n i s removed i n order to s u s t a i n heaving, and i f the s o i l i s f r o s t s u s c e p t i b l e i . e. a s o i l which supports i c e l e n s growth ( 9 6 ) .  Fundamental p r i n c i p l e s i n v o l v e d i n heaving have been reported  e a r l i e r by Taber  (l32),  Bouyoucos and McCool  more r e c e n t l y by Penner ( 9 7 , 9 8 , 9 9 , 100).  ( 2 l )  and Munichsdorfer  (91)  and  Serious heaving occurs mainly  d u r i n g periods of l i m i t e d snow cover and f l u c t u a t i n g s o i l and a i r temperatures, p a r t i c u l a r l y when these v a r i a t i o n s are near the f r e e z i n g p o i n t . f r e e z i n g and thawing favour heaving because  Alternate  these c o n d i t i o n s a l s o favour the  accumulation of water near or at the s o i l surface (41, 5 7 , 6 8 ) .  Penner ( 9 6 )  on the other hand has provided convincing evidence that a l t e r n a t i n g f r e e z i n g and thawing, although more damaging to p l a n t s , i s not a requirement f o r f r o s t heaving.  A study of heaving of forage p l a n t s was (.14.)..  conducted by B i s w e l l et a l .  In,general heaving was more, severe to .tap-rooted legumes than to  f i b r o u s - r o o t e d grasses. a l f a l f a was  Holmes and Robertson  (5.7) observed that heaving of  a s s o c i a t e d with c o n d i t i o n s of p a r t i a l l y bare s o i l and that snow  coyer had a h i g h l y p r o t e c t i v e e f f e c t . . Decker and Roniringen ( 4l) found that :  heaving decreased as the amount and u n i f o r m i t y o f . p l a n t coyer increased, with v i r t u a l l y no heaving o c c u r r i n g i n a Kentucky bluegrass sod.  1.2.3.2.  Heaving due to n e e d l e - i c e formation.  Needle-ice i s ground i c e which.takes  the form of f i n e n e e d l e - l i k e  c r y s t a l s i n compact, c l u s t e r s , at or immediately beneath the ground surface and the i c e columns with v e r t i c a l v o i d s between them, standing upright perpendicular to the c o o l i n g surface {47.).  I t occurs i n f i n e - g r a i n e d ,  bare or s p a r s e l y vegetated s o i l s with no snow cover, and i s formed by the abrupt s u r f i c i a l  f r e e z i n g of s o i l i n i t i a l l y completely f r e e of f r o s t .  i c e needles l i f t  a t h i n l a y e r of s o i l or pebbles, so that the ground surface  appears q u i t e normal except f o r numerous small holes i n the c r u s t . ice  The  Needle-  i s formed at the.contact of a s u p e r f i c i a l l y f r o z e n s o i l l a y e r with moist  unfrozen s o i l beneath.  Water i s drawn from the unfrozen s o i P s pores to  form the c r y s t a l s ('113, 127).  Needle-ice i s best formed when the  drops J u s t a few degrees below freezing..  temperature  I f the temperature gradient  between a i r and s o i l . i s very steep, f r o s t p e n e t r a t i o n i s so r a p i d that no water-segregating and ice-column generating zone 1  can be e s t a b l i s h e d .  these circumstances the s o i l f r e e z e s homogeneously .(13)..  In  When the i c e melts,  the s o i l s e t t l e s back to n e a r l y . i t s o r i g i n a l l e v e l with a noticeable,, accumulation of moisture drawn to the surface by the growing needles.  The  .  process of needle-ice formation may  9  repeat night a f t e r night i f moisture and  temperature c o n d i t i o n s are f a v o r a b l e .  Thus, needle-ice i s common i n milder  temperate c l i m a t e s .  E a r l y observations on needle-ice were made i n Southern  Germany by Mohl  and Sachs  (89)  i s T r o l l ( 1 3 6 ) who  (l09 ) :  i n 1860.  Notable a l s o f o r h i s c o n t r i b u t i o n s  t r a v e l l e d e x t e n s i v e l y observing a l l types of f r o s t  effects  on s o i l s and described the f r o s t climates of the world. Needle-ice may  be very damaging through i t s mechanical a c t i o n on  young, t h i n l y seeded p l a n t s (-24, 5 1 , 5 2 ) ;  . E s p e c i a l l y i n areas such as South--  western B r i t i s h Columbia, where i t i s a frequent p r a c t i c e to e s t a b l i s h t u r f grasses and forage i n the f a l l , needle-ice i n j u r y i s common, e s p e c i a l l y since r e p a i r of plant t i s s u e s during the winter months i s slow ( 2 4 ) . • The mechanism of heaving  of seedlings has been described by Schramm; ( 1 1 3 )  a t h i n surface of the wet  s o i l f r e e z e s , the s e e d l i n g shoots become  f i r m l y encased i n i t , enabling the elongating i c e columns immediately below to exert an upward t h r u s t .  The s e e d l i n g s , rooted i n completely  s o i l , are e i t h e r p u l l e d upward, or i f rooted too f i r m l y are t o r n i n The process i s analogous to clamping a c o l l a r on the shoot at the surface, against which upward pressure can be exerted.  unfrozen two.  soil  Seedlings s u f f e r  extensive damage when t h i s process repeats i t s e l f f o r a number of n i g h t s .  1.2.4.  Winter Drought I n j u r y  During a sudden warm period i n winter, when the s o i l i s s t i l l f r o z e n , p l a n t s have great d i f f i c u l t y i n r e p l a c i n g water l o s t by the shoots i n t r a n s p i r a t i o n . This c o n d i t i o n i s due to a l a g i n temperature change of the s o i l with a change i n the atmospheric temperature ( 9 4 , 1 2 2 , 1 2 3 , 1 4 1 ) , Kramer  (66),  Polunin  (103),  and Wilner  (l49)  reported that the cause  10  of decreased water absorption by plants at low s o i l temperature i s the combined e f f e c t of decreased permeability of the root membranes and increased v i s c o s i t y o f the water i t s e l f .  T h i s r e s u l t s i n increased r e s i s t a n c e to water  movement across the l i v i n g c e l l s o f the r o o t s .  High winds, and e s p e c i a l l y  warm breezes that break a period o f c o l d weather, have strong d r y i n g e f f e c t s . Evergreen p l a n t s are p a r t i c u l a r l y s u s c e p t i b l e to t h i s i n d i r e c t type of low temperature i n j u r y which has been a p t l y c a l l e d parch b l i g h t ( 9 0 ) .  Their  f o l i a g e i s commonly observed t o t u r n brown i n l a t e winter or s p r i n g a f t e r desiccation.  1.2.5.  Lesions  When a i r temperatures drop r a p i d l y the outer l a y e r s o f a plant stem o r trunk c o o l f a s t e r than the i n n e r p a r t s .  The r e s u l t a n t tension i s  at times s u f f i c i e n t to- cause the stem to crack open (40). has been reported as e a r l y as 1683 hy Bobart ( l 6 ) .  T h i s phenomenon  Such l e s i o n s may break  open i n tree trunks accompanied by a loud c r a c k i n g n o i s e .  The crowns of  p e r e n n i a l forbs such as a l f a l f a s u f f e r s i m i l a r i n j u r y , and the damage i s u s u a l l y aggravated by the- entrance of p a r a s i t i c f u n g i through these l e s i o n s . S i m i l a r tensions develop, i n the outer l a y e r s o f tree trunks, during c o l d sunny weather.  The side of. the trunk exposed t o the sun's rays i s warmed,  while the shaded side remains a t the ambient temperature.  Tensions developed  as a r e s u l t o f the temperature d i f f e r e n t i a l may cause sloughing  of the bark,  i  k i l l i n g the tree ( 4 8 ) .  1.2.6.  A l t e r n a t i n g Temperatures  A l t e r n a t i n g temperatures above and below f r e e z i n g , are harmful,  11  e s p e c i a l l y when there i s no snow cover.  Warm periods can r e s u l t i n a break  i n the dormancy •witli. a r e d u c t i o n of f r o s t r e s i s t a n c e as p l a n t s respond to higher temperatures and are unable to reharden  s a t i s f a c t o r i l y or q u i c k l y  enough upon exposure to a drop i n temperature ( 1 3 , 1 9 ) .  The extent of  damage depends to a considerable- degree on the r a p i d i t y of temperature alternation. concluded  Dexter ( 4 3 ) c a r r i e d out a s e r i e s of experiments on a l f a l f a ,  and  that the p l a n t s could reharden i f growth had not occurred to any  great extent and provided s u f f i c i e n t carbohydrate  reserves were a v a i l a b l e .  would thus appear that a l e s s e r degree of f r o s t r e s i s t a n c e would be  It  developed  f o l l o w i n g each period of "dehardening."  1.2.7.  Ice-Sheet  I n j u r y or Smothering  Overwintering herbaceous p l a n t s , such as forages, may  be i n j u r e d  when covered f o r long periods i n . a continuous ice-sheet covering l a r g e t r a c t s of land, u s u a l l y over unfrozen or only p a r t i a l l y f r o z e n s o i l ( 5 0 , 1 2 3 ) .  Ice-  sheets commonly form where drainage i s poor and water; c o l l e c t s during winter, as a r e s u l t of changing temperatures with warm periods accompanied by r a i n or snow-melt.  The accumulated water f r e e z e s to a s o l i d l a y e r of i c e during sub-  sequent cold p e r i o d s .  Ice-sheets form l e s s f r e q u e n t l y as a r e s u l t of f r e e z i n g  rainstorms, and when t h i s occurs, i c e may topography  (l23).  cover everything regardless of  Ice-sheets are o f t e n a s s o c i a t e d with a l t e r n a t e f r e e z i n g  and thawing with i n t e r m i t t e n t snow;, f r e q u e n t l y the i c e and snow c r u s t s be s e v e r a l inches i n thickness and moreover may months.  may  p e r s i s t f o r many weeks or  Damage to forage- crops due to i c e - s h e e t s i s sporadic but often wide-  spread and devastating, e s p e c i a l l y i n areas such as the North C e n t r a l States of the United States, i n adjacent Canada, i n Eastern and Northern Europe, f o r an example, i n Holland and the c o a s t a l regions of Norway ( 1 1 9 ' ) .  12 1.3.  OPINIONS ON THE CAUSE OP ICE-SHEET DAMAGE TO PLANTS  Observations- on i c e - s h e e t i n j u r y i n the f i e l d are complicated by many damaging f a c t o r s which occur during the winter; d i f f i c u l t to evaluate the r e a l  1.5.1.  i t i s therefore, often  cause.  Evidence Against Smothering as a Cause of Ice-Sheet In.jury  Jones (62) c a r r i e d out a d e t a i l e d h i s t o l o g i c a l examination alfalfa  on  p l a n t s c o l l e c t e d from f i e l d s i n Southern Wisconsin which had been  covered by heavy i c e - s h e e t s so l o n g that much of the stand was k i l l e d . found that i n j u r y was  always a s s o c i a t e d with r i f t i n g of c e l l s ,  n e c e s s a r i l y as a consequence of t h e i r mechanical separation.  although not Jones  (62)  removed a number of p l a n t s from beneath an i c e - s h e e t and sectioned them. found the t i s s u e almost completely d i s o r g a n i z e d ;  r i f t s extended  though coherent.  separated  of the•cambium r e g i o n were c o l l a p s e d ,  In some p l a n t s n e a r l y a l l of the t i s s u e except the cambium  was d i s o r g a n i z e d , while i n others r i f t s were found only along the rays. et a l . ( 2 2 )  He  the e n t i r e  length of the rays, the l a r g e r groups of parenchymatous c e l l s had i n t o loose aggregations, and the c e l l s  He  Brierley  c a r r i e d out a number of experiments, both i n the f i e l d and with potted  strawberry p l a n t s .  Although they found that i c e or waterlogged  frozen s o i l i s  quite impermeable to gases, and that carbon d i o x i d e d i d accumulate to l e v e l s as high as 24$ i n the-atmosphere surrounding p l a n t s overwintering under such c o n d i t i o n s , they concluded that i f the temperature  was kept above the point  of i n j u r y , an i c e s e a l maintained f o r as long as 10 weeks was not i n i t s e l f a cause of i n j u r y to strawberry p l a n t s .  They suggested that much of the  i n j u r y i n the f i e l d , commonly a t t r i b u t e d to "ice-smothering,"  1  i s due to l e t h a l  15 temperatures, excess water at the time the p l a n t s emerge from the dormant c o n d i t i o n , or disease that may as broken r o o t s .  i t s i n c e p t i o n i n winter l e s i o n s such  More r e c e n t l y Beard and O l i e n (9)  annual bluegrass, which was than 60 days, was  have had  i n d i c a t e d that death of  covered by heavy ice-sheets i n the f i e l d f o r more  caused by d i s r u p t i o n of t i s s u e o r g a n i z a t i o n accompanied by  d e s t r u c t i o n of p r o t o p l a s t s i n roots and e s p e c i a l l y the crown t i s s u e f o l l o w i n g i n t e r c e l l u l a r i c e c r y s t a l formation.  In l a t e r work Beard (6, 7,  8)  subjected grasses to v a r i o u s types of i c e - c o v e r s , both i n the f i e l d and under controlled-environmental occurred when a s l u s h was  conditions.  In the f i e l d the most s i g n i f i c a n t i n j u r y  created which was  compacted and then f r o z e n .  suggested that such conditions could r e s u l t i n an increased hydration i n the t i s s u e due Increased  (7)  level  to increased contact between the plant t i s s u e and the s l u s h .  t i s s u e moisture content r a i s e s the k i l l i n g temperature with mechanical  d e s t r u c t i o n r e s u l t i n g ' f r o m the formation experimental c o n d i t i o n s he found that  days ( 6 ) .  Under  Some v a r i e t i e s survived f o r as  The most severe i n j u r y r e s u l t e d from the storage  plugs i n i c e b l o c k s . and Beard (8.)  of l a r g e i c e masses.  Toronto'- creeping bentgrass e x h i b i t e d  a high degree of tolerance to i c e - c o v e r s . as 120  Beard  long  of sod  F r e e z i n g such plugs i n v o l v e d t h e i r submergence i n water  feels- that an abnormally high-moisture content may  have developed  i n the crown t i s s u e of the p l a n t , thus, •leading to complete c e l l u l a r d i s r u p t i o n at  the time of f r e e z i n g .  The  r e s u l t s of t h i s treatment suggest that combina-  t i o n s of f r e e z i n g and thawing-possibly moisture l e v e l s may  i n a s s o c i a t i o n with high plant t i s s u e  be of g r e a t e r s i g n i f i c a n c e i n winter i n j u r y associated  with ice-sheets than d i r e c t oxygen s u f f o c a t i o n or t o x i c accumulations. Brink (23)  and more r e c e n t l y Sjoseth (H9)  s i m i l a r l y found v a r i e t a l  d i f f e r e n c e s i n the a b i l i t y of a l f a l f a , red c l o v e r and timothy to withstand ice-encasement, but had no suggestions as to the mechanism of ice-sheet damage.  14 V a s i l ' y e v (l39)  found i c e to be quite permeable  to a i r and s t a t e s that  p e r e n n i a l grasses w i n t e r i n g under i c e do not d i e no matter how remain that way, under snow.  l o n g they  and commence t h e i r growth i n the s p r i n g l i k e p l a n t s w i n t e r i n g  According to V a s i l ' y e v (139)  c u l t i v a t e d p l a n t s react the same way  on f i e l d s covered with i c e a l o n g time,  1.3.2.  Evidence Supporting Smothering as a Cause of lee-Sheet In.iury  In contrast to V a s i l ' y e v , Hemmingsen (56)  of the I n s t i t u t e of  Zoophysiology.in Oslo, Norway, found carbon dioxide permeation through i c e to be about two m i l l i o n times l e s s than i n water, and no permeation of oxygen through i c e could be detected.  In sea i c e gas permeates through i n t e r c r y s t a l l i n e brime  f i l m s r a t h e r than through i c e c r y s t a l s .  In asking the question whether or  not gas exchange can p o s s i b l y take place- through i c e which entombs organisms, Scholander and a s s o c i a t e s ( l l 2 ) found that i c e - i s extremely gas t i g h t , indeed ;  as t i g h t as i s g l a s s to helium.  An o l d and w e l l e s t a b l i s h e d f a c t i s that f i s h  are k i l l e d by s u f f o c a t i o n ' i n l a k e s that are f r o z e n over f o r longer periods of time (144, 53)•-  Bergman (lOJ) found that water under i c e i n cranberry bogs  to be d e f i c i e n t i n oxygen.  Bugaevskii and Z i t n i k o v a (28) have observed that  winter wheat p l a n t s beneath an i c e crust at -1.0° to -9.0°C began to decrease i n v i g o r a f t e r the twenty-third day and i n f i f t y - f o u r days a l l were dead. They suggest a l a c k of oxygen as a p o s s i b l e cause of death.  Similarly  Tumanov (l35) found i c e to be impermeable to a i r and suggested that p l a n t s s u f f o c a t e under i c e . Graber (49, 50). s t a t e d that an ice-sheet causes damage l a r g e l y -by i t s d u r a t i o n and that death or i n j u r y to the sealed plants seems to r e s u l t from the accumulations of by-products from r e s p i r a t i o n .  A few years  l a t e r Sprague and Graber (125? 126) c a r r i e d out a s e r i e s of experiments.  They  !5 trimmed down a l f a l f a p l a n t s and stored them a t r-3.5°C i n v a r i o u s media, such as carbon d i o x i d e , n i t r o g e n , water with d i f f e r e n t l e v e l s o f carbon d i o x i d e , and stored them a l s o e n t i r e l y encased i n i c e .  They found that o f a l l the  storage treatments, the p l a n t s kept i n water, e s p e c i a l l y saturated with carbon dioxide and those f r o z e n i n d i r e c t contact with i c e to have s u f f e r e d the greatest damage. and  They concluded that-the i c e i n h i b i t s the removal, escape,  e x t e r n a l d i f f u s i o n of carbon dioxide and other products of aerobic and  anaerobic  r e s p i r a t i o n . The e x c l u s i o n o f oxygen d i d not seem to be an immediate  cause of i n j u r y o r death.  Before the absence o f oxygen became a l e t h a l  f a c t o r , i t appears that concentration o f carbon dioxide and other r e s p i r a t o r y products r e s u l t i n g from .the^encasement of the p l a n t s i n i c e , became damaging. Sprague and Graber (l25) made measurements o f carbon dioxide l i b e r a t e d by dormant a l f a l f a p l a n t s stored i n closed t e s t tubes of a i r o r o f n i t r o g e n , and  showed that a d i r e c t r e l a t i o n s h i p e x i s t e d between the developing  t r a t i o n s of the gas and the i n j u r y sustained.  concen-  They c i r c u l a t e d atmospheres of  2 5 $ and 5C$ carbon dioxide and found' t h a t e x t e r n a l accumulations of carbon d i o x i d e are not d i r e c t l y t o x i c , but that a d u r a t i o n of exposure to the gas may cause i n t e r n a l concentrations the c e l l s .  o f r e s p i r a t o r y compounds which are t o x i c to  Dale Smith ( l 2 0 , 12l) c a r r i e d out experiments to determine the  s u r v i v a l o f winter-hardened legumes encased i n i c e .  He found that carbon  dioxide l i b e r a t e d by equal weights o f l a d i n o and common white c l o v e r s t o l o n s stored i n sealed t e s t tubes a t 0 ° C showed that the carbon d i o x i d e accumulat i o n s were c o n s i s t e n t l y • h i g h e r f o r the l a d i n o c l o v e r .  The more severe i n j u r y  to the l a d i n o c l o v e r under the sealed c o n d i t i o n s o f i c e encasement appears to be a s s o c i a t e d with the l a r g e stolons-and-the concentrations  and pressures' of carbon'dioxide  accumulation of high i n the surrounding  media.  Dale. Smith ( l 2 l ) 'also' pointed i o u t ^ t h a l ; : t h e ' a b i l i t y o f winter-dormant legumes 1  16 to withstand i c e encasement when the temperature was s u r v i v a l , was  i n some manner r e l a t e d to t h e i r inherent c o l d hardiness,  that t h i s r e l a t i o n s h i p i s associated maintained d u r i n g winter dormancy.  and  with the l e v e l of metabolic a c t i v i t y The  l e s s hardy-legumes maintain a higher  l e v e l of a c t i v i t y than do the more hardy legumes. differences  not a d i r e c t f a c t o r i n  He  suggests that  i n s u r v i v a l under the sealed c o n d i t i o n s of i c e may  from the by-products of r e s p i r a t i o n , such as carbon dioxide, i n j u r i o u s concentrations more r a p i d l y i n and  the  r e s u l t i n part  which may  reach  around the sealed plant parts of  the l e s s hardy legumes than i n the case of the more hardy types.  1.4.  RELEVANT SOIL ATMOSPHERE STUDIES  Gaseous exchange between the s o i l atmosphere and  the a e r i a l  atmosphere i s p r i m a r i l y accomplished by d i f f u s i o n (5, 63, 106,  107)  and  to  a l e s s e r extent by m e t e o r o l o g i c a l f a c t o r s such as s o i l temperature changes, barometer v a r i a t i o n s , a c t i o n of the wind and  changes i n the amount of pore  space occupied by a i r as a r e s u l t of the entrance of water ( 5 ) .  Most of  the  •1.  work to date d e a l i n g with d i f f u s i o n i n the s o i l has i n the gaseous phase alone.  L i t t l e a t t e n t i o n has  concentrated on d i f f u s i o n  been given to the  i n f l u e n c i n g oxygen d i f f u s i o n i n the immediate root environment. between atmospheric oxygen and  the oxygen -supply to the plant  The  factors link  roots i s b e l i e v e d  to be the s o i l s o l u t i o n , or more p r e c i s e l y , a t h i n f i l m of water covering surface of the r o o t s .  The  from the s o i l atmosphere. investigators aeration.  The  the  oxygen of the s o i l s o l u t i o n , i n turn, i s replenished Buckingham' (2?) i n 1904  was  one  of the  first  to apply the k i n e t i c theory of the d i f f u s i o n of gases to importance of d i f f u s i o n i n s o i l a e r a t i o n was  a t t e n t i o n more r e c e n t l y by Penman ( l O l , 102),  soil  given renewed  Blake and Page ( 1 5 ) , T a y l o r (l33)>  17  Van Bavel (3, 4 ) , and C u r r i e (35, 36, 31, 38, 39). The presence o f an impermeable i c e l a y e r on the surface o f the s o i l w i l l exclude a l l gas exchange with the atmosphere.  The s o i l under an ice-sheet  i s u s u a l l y wet o r even saturated and unfrozen o r f r o z e n only a few centimeters deep.  Under such moist c o n d i t i o n s , the general p a t t e r n of a decrease i n the  oxygen concentration accompanied by an i n c r e a s e i n the carbon dioxide concentration does not n e c e s s a r i l y hold.  O c c a s i o n a l l y , as Russel and Appleyard  (108) and Boyntan and Compton ( l 9 ) have shown, the contents o f both oxygen and carbon dioxide may r i s e and f a l l together, and when the s o i l contains much water the oxygen content may f a l l - by more than the carbon dioxide rises.  content  This i s probably due to•the s o l u b i l i t y o f carbon dioxide i n water being  much greater than that of oxygen.  Thus, i n wet s o i l s an appreciable amount  of carbon dioxide i s present i n s o l u t i o n i n the s o i l water, but Russel and Appleyard  (l08) could not detect any oxygen i n s o l u t i o n , a r e s u l t which they  a t t r i b u t e d to the immediate u t i l i z a t i o n o f any d i s s o l v e d oxygen by s o i l microorganisms.  Scott and Evans ( l l 5 ) made a study of d i s s o l v e d oxygen i n saturated  s o i l , and found that, i n the s o i l s studied, a l l of the oxygen added to an a i r dry s o i l by f l o o d i n g i t with a i r - s a t u r a t e d d i s t i l l e d water disappeared 10 hours.  within  A f t e r the oxygen was depleted, more oxygen was added to the same  saturated s o i l s by f l u s h i n g them with oxygen laden s o l u t i o n s . oxygen disappeared  T h i s added  even more r a p i d l y than i t d i d i n f r e s h l y flooded s o i l s .  Scott and Evans (115) d i d not make any c l e a r separations of the m i c r o b i a l a c t i v i t y and chemical demands f o r oxygen, however, they d i d observe that the rate of oxygen consumption i s g r e a t l y reduced i f the s o i l i s s t e r i l i z e d .  The  f i n d i n g s of Scott and Evans have been more r e c e n t l y substantiated by the work of Yurkevich e t d . (152') who found 'that the water i n r a i s e d , t r a n s i t i o n a l - a n d fen bogs d i d not contain any d i s s o l v e d oxygen, while the carbon dioxide concentration was c o n s i d e r a b l e .  18 The e a r l i e r l i t e r a t u r e on p l a n t responses t o s o i l a e r a t i o n has been reviewed by Cannon and Free (30) and Clements ( 3 2 ) .  The broad aspect of  the r e l a t i o n s h i p of the p l a n t to the gaseous composition of i t s environment has been reviewed i n d e t a i l by S t i l e s (128). thoroughly  Turner ( l 3 8 ) has r e c e n t l y  reviewed the l i t e r a t u r e p e r t a i n i n g to the p h y s i o l o g i c a l response of  p l a n t s to low oxygen t e n s i o n s or a n a e r o b i o s i s .  Extensive s t u d i e s on root  r e s p i r a t i o n have been c a r r i e d out by Berry and N o r r i s ( l l , 12), Zimmerman and Berry (153), and Zimmerman, Berry and Crenshaw ( l 5 4 ) . out on onion r o o t s .  T h e i r s t u d i e s were c a r r i e d  Although the v e l o c i t y of oxygen consumption i n d i f f e r e n t  segments of root at d i f f e r e n t temperatures as a f u n c t i o n of p a r t i a l  pressure  of oxygen was measured, the lowest temperature used i n the study was  15  c  (11,12).  During the anaerobic p e r i o d i n the s t u d i e s of the r e s p i r a t o r y rebound Zimmerman et a l . ( l 5 4 ) observed t h a t the r o o t s became " l e a k y " w i t h time.  Some unknown  substance was g i v e n o f f by the roots.- Wanner ( l 4 3 ) observed a s i m i l a r phenomenon and demonstrated t h a t the accumulation of t h i s unknown m a t e r i a l suppressed the metabolic uptake of oxygen even though carbon d i o x i d e was removed.  Wanner's f i n d i n g s i n d i c a t e , however, t h a t oxygen d i f f u s i o n i s the  f i r s t l i m i t i n g f a c t o r i n root r e s p i r a t i o n , and that accumulation of carbon d i o x i d e and other root:by-products plays a secondary r o l e .  Letey et a l . (76)  s t u d i e d the e f f e c t of temperature on oxygen d i f f u s i o n r a t e s and subsequent shoot growth, root>growth, and m i n e r a l content of two p l a n t s p e c i e s .  They  found t h a t not only d i d the r a t e of root r e s p i r a t i o n vary w i t h temperature, but a l s o w i t h the supply of oxygen, since w i t h i n c r e a s i n g temperature the s o l u b i l i t y of oxygen,decreases,.'-.while the..diffusio'nL'coefficient through both gas and l i q u i d i n c r e a s e s .  More- r e c e n t l y Lemon (72) and Wiegand  and K r i s t e n s e n and Lemon (67) i n t h e i r s e r i e s of a r t i c l e s on the  (73) subject  of s o i l a e r a t i o n and p l a n t root r e l a t i o n s , t h e o r e t i c a l l y considered  19 the root oxygen "demand" aspects and the "supply" c h a r a c t e r i s t i c s o f the s o i l environment.  According t o Lemon and Wiegand (73) the f o l l o w i n g f a c t o r s  determine these "demand" c h a r a c t e r i s t i c s : (a)  The r a t e o f metabolic oxygen uptake by root t i s s u e s v a r i e s w i t h the g e n e t i c background and the p h y s i o l o g i c a l age o f the t i s s u e .  (b)  When oxygen i s p l e n t i f u l , the " s u b s t r a t e " supply a t the r e a c t i o n l o c i determines the r e a c t i o n r a t e . • Chemical processes i n v o l v e d i n " s u b s t r a t e " supply are p a r t i c u l a r l y s e n s i t i v e t o temperature.  (c)  When the oxygen c o n c e n t r a t i o n a t the root surface i s below the c r i t i c a l l e v e l , d i f f u s i o n c o n t r o l s the r a t e o f oxygen uptake.  This  p h y s i c a l process i s r e l a t i v e l y i n s e n s i t i v e t o temperature. (d)  The c r i t i c a l oxygen c o n c e n t r a t i o n a t the root surface i s s t r o n g l y dependent upon the r a d i u s o f the r o o t and the d i f f u s i o n c o e f f i c i e n t of oxygen w i t h i n the r o o t . I n the f i n a l a r t i c l e o f the s e r i e s K r i s t e n s e n and Lemon (67)  i n t e g r a t e d the "demand" and "supply" aspects o f oxygen i n the root s o i l system, demonstrating  t h a t the two aspects are interdependent.  S t u d i e s on the oxygen requirement o f crop r o o t s and s o i l s under f i e l d c o n d i t i o n s have been c a r r i e d out r e c e n t l y by Brown, Fountaine and Holden (26). These s t u d i e s were c a r r i e d out d u r i n g the growing season, and as y e t , no i n f o r m a t i o n i s a v a i l a b l e on the oxygen requirement o f crop r o o t s and s o i l s d u r i n g the dormant w i n t e r season.  Numerous a r t i c l e s have d e a l t w i t h e f f e c t s  of inadequate s o i l a e r a t i o n on crop p l a n t s (54, 75, 77, 78, 129, 147). I n s t u d i e s o f s o i l a e r a t i o n , oxygen has r e c e i v e d the major emphasis, w h i l e f a r l e s s data i s a v a i l a b l e on the e f f e c t s o f s o i l carbon d i o x i d e on p l a n t s . Since the a v a i l a b l e i n f o r m a t i o n has been thoroughly reviewed by S t i l e s ( l 2 8 ) and Turner. ( l 3 8 ) o n l y b r i e f reference w i l l be made here.  Germination,  t o t a l plant  20 growth, and root growth are u s u a l l y i n h i b i t e d by increased concentrations carbon d i o x i d e (32,  64).  of  The l e v e l of carbon d i o x i d e causing r e v e r s i b l e and  i r r e v e r s i b l e i n h i b i t i o n i s q u i t e v a r i a b l e and d i f f e r e n t responses are e x h i b i t e d by v a r i o u s p l a n t s (30, 55, 65, 74, 93, 140).  Clements (32)  recognized  that  carbon d i o x i d e l e v e l s from 2 to 20ft> would cause i n j u r y to p l a n t s w h i l e 5 to 1C$> carbon d i o x i d e i s f r e q u e n t l y encountered i n the s o i l and concluded t h a t i n j u r y from carbon d i o x i d e i s more frequent than commonly supposed. S t o l w i j k and Thimann (130) was  showed t h a t growth of r o o t s of c e r t a i n crop p l a n t s  completely i n h i b i t e d i f the root medium was aerated w i t h 6.5^  carbon  d i o x i d e i n a i r , w h i l e c e r t a i n g r a i n crops were u n a f f e c t e d by such a  treatment.  N o r r i s , Wiegand and Johanson (92) made a study of the e f f e c t of carbon d i o x i d e on r e s p i r a t i o n of e x c i s e d onion root t i p s i n high-oxygen atmospheres, and found that carbon d i o x i d e causes a depression of gas exchange, and t h a t the degree of depression i s r e l a t e d to the c o n c e n t r a t i o n of carbon d i o x i d e One  present.  f u r t h e r aspect which deserves some c o n s i d e r a t i o n i s the p o s s i b l e  t r a n s p o r t of atmospheric oxygen through p l a n t p a r t s or stubble p r o t r u d i n g through an i c e - s h e e t to the root system.  Many workers have demonstrated t h a t  the r o o t s of hydrophytes depend on the a e r i a l p a r t s f o r t h e i r oxygen supply, ( l , 34, 146.). The r o o t s u t i l i z e oxygen which d i f f u s e s from the shoot through the i n t e r c e l l u l a r space system.  More r e c e n t l y , e s p e c i a l l y w i t h the  use of " l a b e l e d " oxygen, i t has been demonstrated t h a t oxygen w i l l d i f f u s e down to the r o o t s of non-aquatic p l a n t s , such as b a r l e y , grown i n anaerobic s o l u t i o n s (31, 105, 124).  Once again the s t u d i e s were c a r r i e d out w i t h  a c t i v e l y growing p l a n t s , and no i n f o r m a t i o n i s a v a i l a b l e on the d i f f u s i o n of gases i n dormant o v e r w i n t e r i n g p l a n t s , or dead s t u b b l e .  21  2.  MATERIALS AND METHODS  To determine the nature of ice-sheet damage to overwintering p l a n t s , an attempt was made to simulate  forage  as c l o s e l y as p o s s i b l e , i n a c o l d  room, c o n d i t i o n s found i n the f i e l d , with one exception being that the plants would not be exposed to low temperature extremes as may occur i n areas s u s c e p t i b l e to winter i c e - s h e e t s .  The experiments were, t h e r e f o r e , planned  to eliminate d i r e c t i n j u r y t o the p l a n t s from low temperatures so that the i n f l u e n c e s o f the gases i n the ice-sheet covered s o i l could be a s c e r t a i n e d .  2.1.  BOXES WITH TEMPERATURE CONTROL AND POTS  To simulate n a t u r a l ice-sheet c o n d i t i o n s , boxes with temperature c o n t r o l , were constructed,  not u n l i k e the f r e e z i n g box f o r plant  s t u d i e s designed by Peake ( 9 5 ) .  hardiness  The box as i l l u s t r a t e d i n F i g . 2.1 was  2 2 ... constructed of h a l f - i n c h plywood i n s u l a t e d w i t h g l a s s wool on a l l s i x inner; 1  side's.  A 15'. watt, l i g h t :bulb. mounted' i n the center- of the box,: and connected  to a rheostat oh the- outside j'maintained a temperature i n s i d e the box at. approximately 1°C. , Pour potted p l a n t s were mounted i n h o l e s cut i n t o t h e ' removable'lid.. F o r complete i n s u l a t i o n strips.">of i n s u l a t i n g m a t e r i a l were wrapped around: the -upper side of the pot adjacent to the i n n e r side of the lid). ( F i g , 2.2).. . Caulking .compound was a p p l i e d between, the rim of the pots and the.plywood, lid,.- so that on flooding.the surface no- water would, leak i n t o the box.- . Pots-used i n t h i s study,: a t f i r s t were "Beacon"'polyethylene waste • baskets, 25 cm..high and-with an i n t e r n a l diameter of "19 cm..  To 'ensure that  the. pots were gas t i g h t , three coatings of tygon paint were a p p l i e d to the i n n e r * a n d outer surfaces.- To t e s t t h e i r permeability,, a painted pot,- w i t h ; , i t s top. sealed and with .carbon d i b x i d e - f r e e a i r inside,-, was placed i n a chamber ;  of carbon'dioxide atmosphere ( F i g . 2.4). '* Gas- samples were withdrawn p e r i o d i c a l l y from the pot through,a sampling tube sealed with a s i l i c o n e rubber septum.  The . :  carbon dioxide.was found-to permeate•quite r e a d i l y and w i t h i n 3 weeks i t s l e v e l i n the pot was i n e q u i l i b r i u m with/that i n the outer chamber.-.• Consequently.,; \ a f t e r using, the p l a s t i c : p o t s in-Experiments I and I I , they were s u b s t i t u t e d with a i r — t i g h t metal-.syrup cans -in a l l subsequent  experiments.:  The dimensions of the metal, pots are i n d i c a t e d i n Fig.-.2.3,.  Four  metal .brackets, soldered to the'upper rim'supported the cans when they were mounted, in" the holes, i n the l i d of-' the-, "box. with ^temperature' c o n t r o l . • Two: 1  copper f l u s h i n g tubes., passing through d r i l l e d holes were soldered to the - • containers' :• walls.-. P e r f o r a t e d tygon;tubing, was attached .to-the-copper tubes on the inner,, side, o f the pot to f a c i l i t a t e f l u s h i n g of'the s o i l .  The lower tygon tube,  23 as i n d i c a t e d i n F i g . 2.2,. was  longer than the'.upper,  so that excess water.,  could; r e a d i l y be drained from-the s o i l following'the formation of "an ice-sheet on the' s o i l s u r f a c e .  Rubber tubes, connected  to'"-the copper tubes on the outer ,  side' of the - pot-,; were extended through holes i n ' the ."'-insulated,, w a l l s of the ; box to the outside ( F i g . - 2 . l ) . ' In t h i s way,  a connection was  achieved from.-  the potted s o i l atmosphere to the outside of the box with'temperature  .  control. For ,.ice-sheet s t u d i e s , the boxes with temperature c o n t r o l were moved, i n t o a "walk-in" c o l d room, maintained at approximately -3.O^C.- . The boxes :  were'-arranged on a "Dexion" s t e e l frame as shown i n F i g . 2.3, s t r i p s , 2 cm", wide and 0.5  cm. t h i c k were-nailed- on  adjacent two or. three boxes. of a d j o i n i n g boxes was  t  2.6.  Wooden  a l l the'upper edge's of :  A continuous water t i g h t surface over,a  series  achieved b y c o v e r i n g any voids;with masking tape :  and.  caulking. c o m p o u n d T h u s , with these- edges n a i l e d t o i the upper s i d e s of the' boxes, an. ice-sheet,''covering a s e r i e s of adjacent boxes, c o u l d b e f l o o d i n g the water-tight surface ( F i g . 2.3,  2..2, TEMPERATURE, PRESSURE, AND  formed by-. .  2.6)-.  MOISTURE MEASUREMENT  To vary the' temperature of the atmosphere at• the.,soil surface, an i n s u l a t e d .plywood thawing-lid was-constructed boxes.  so as to. cover two .adjoining ,  The l i d , 15 cm. high, had two.60 watt.light, bulbs mounted on t h e . :  :  t  i n n e r side ( F i g . 2.3).. The two bulb's were connected  to a-rheostat-.with',  which temperature under the thawing-lid could be-varied from -3.0°C ( i . e . ccold room temperature) to +.15.0°C. . Temperatures were measured with' a "'Rubicon" potentiometer which  24 was  connected,  through a r o t a r y switch t o a s e r i e s of 26>gage  thermocouple wires.  copper-constantan  The thermocouple beads were coated with.an,epoxy glue to  prevent corrosion,and were mounted i n v a r i o u s l o c a t i o n s , such as i n the potted s o i l , , inside'and on the surface of the i n s u l a t e d box; beneath the ice-sheet.. To detect; any--pressure.change-that  might occur, i n the potted  under a prolonged ice-sheet cover, manometers were attached to-the f l u s h i n g tubes, while'the upper tubes' were kept, c l o s e d .  soil  lower  The manometers were  adapted from Warburg respirometers and were f i l l e d with manometric f l u i d  (-Fig; 2.7.).  To measure* the-.moisture < i n the s o i l under an i c e - s h e e t , Bouyoucos ; gypsum blocks were placed i n the pots, with the'-leads passing through the i c e ; to  the s u r f a c e .  A l l the moisture blocks were standardized at 0°C.+ 1°C. :  2.3.  PLANT SPECIES AND  VARIETIES  In a l l the experiments,: i n v o l v i n g the boxes with temperature c o n t r o l , f i e l d grown a l f a l f a was used. Western Canadaj  Three v a r i e t i e s of a l f a l f a most.common to  'Ladak', 'Vernal', and  'Dupuits'.'were seeded on June 20,-1965,  i n bands, which were embedded i n the' f i e l d .  Of the three v a r i e t i e s , 'Ladak'-  i s the most winter hardy and i s s u i t a b l e f o r areas such 'as Northern Columbia' and A l b e r t a .  'Vernal' i s f a i r l y winter-hardy-and  i n .the-southern i n t e r i o r of B r i t i s h Columbia.  i s grown e x t e n s i v e l y  .'Dupuits' i s the l e a s t hardy ;  of the three v a r i e t i e s and i n the west i s grown mainly i n the River Valley.  British  lower-Fraser  I r r e s p e c t i v e of t h e i r winter hardiness, the temperatures  used  i n the experiments,: were n o n i n j u r i o u s to a l l three varieties'. Following'germination the a l f a l f a was per band,.  thinned out to a s i n g l e plant  The p l a n t s were then transplanted from the f i e l d i n t o pots and cut  25 to, a height of 6. to.8 cm. a. week p r i o r t o being-covered i n i c e . Since a l l the :  i c e - s h e e t experiments were c a r r i e d out i n the., winter,- the a l f a l f a used i n the-.. study had,been f i e l d hardened.. Vancouver w i n t e r s , however, are:not, v e r y c o l d , so t h a t the p o t t e d . p l a n t s , . a f t e r b e i n g mounted- in'the-boxes'with temperature c o n t r o l , were kept.-for;7 days ,in a -room< maintained, at, 0°C. (+ 1°G) f o r f u r t h e r hardening..; ,  •2.4.  SOIL AND  SOIL ATMOSPHERE- SAMPLING TUBES  A f t e r the p l a n t s had been t r a n s p l a n t e d into, the p o t s , they were. then, weighed j and s o i l - w a s e i t h e r added o r ••.removed, so t h a t - a l l the pots, w i t h i h . each experiment, contained, approximately '-equal q u a n t i t i e s of s o i l . The f i e l d s o i l used-was N i c h o l s o n (Alderwood) a c i d brown-wood.ed sandy loam, w i t h a, pH of approximately 6.Q.. ;  The average organic carbon, content  determined by the -Walkley - . B l a c k method (142) was-found, t o be 7.4$.  F o r an  approximate measurement of the s o i l ' p o r o s i t y , - . c o r e s were taken from the pots u s i n g s m a l l metal cylinders.'.- T o t a l per cent p o r o s i t y .was determined using' the. standard equations T o t a l fo porosity: = ( 1 - Bulk d e n s i t y ) x.ulOO [Particle': density" :  The p a r t i c l e density.-of' the s o i l .was 2.65, w h i l e the p o r o s i t y was found/,to ' be  67$. • S o i l atmosphere-sampling-tubes-were placed i n . t h e s o i l , a s i n d i c a t e d  in. Fig ,2.2. %  The. tubes used, i n t h e experiments,, .-are a m o d i f i c a t i o n of•' a r  design-reported by'Yamkguchi et.-.ali> ,',('151.).  The sampling;tubes,'were  constructed  26 by g l u i n g a gas-chromatograph s i l i c o n e septum t o one end of a g l a s s tube 18 cm. l o n g and w i t h an i n t e r n a l diameter of 5 nim.  The s i l i c o n e rubber  septum was attached w i t h an epoxy g l u e , and to ensure a g a s - t i g h t f i t , as w e l l as to p r o t e c t the septum, a short piece of tygon t u b i n g p r o t r u d i n g a few :  m i l l i m e t e r s above the septum was attached to the end ( F i g . 2.2). experiments,  i t was found,that  In preliminary  the' composition of the gas sampled from the  modified tube was the same as t h a t of the sample taken from the same potted s o i l , u s i n g the tube d e s c r i b e d by Yamaguchi et a l . ( l 5 l ) •  2.5.  SIMULATION OF ICE-SHEETS  On the e i g h t h day i n the n e a r - f r e e z i n g temperature room, (0°C chipped i c e was packed on the surface of the s o i l to a depth of 3 cm.,  +, 1°C)  approximately  and the pots, mounted i n t h e i r boxes, were moved i n t o the -3.0°C room,  where they were arranged  on the Dexion s t e e l frame ( F i g . 2.3,  2.6).  To form an i c e - s h e e t on the s u r f a c e , normally r e q u i r e d 3 days. :  At f i r s t the surface l a y e r of s o i l under the chipped i c e was permitted t o f r e e z e f o r 6 hours to form a t h i n f r o z e n - c r u s t . . F o l l o w i n g t h i s i n i t i a l p e r i o d , - f r e e z i n g water was l i g h t l y s p r i n k l e d on.the surface and allowed t o freeze.  T h i s process was repeated' every few hours f o r 2 days, a f t e r which  the. l i g h t bulbs i n the boxes were switched on t o prevent the s o i l from f r e e z i n g too: deeply.  Once a d e f i n i t e ice-rsheet ;had been formed, h e a v i e r doses of  f r e e z i n g water c o u l d be a p p l i e d to the surface without danger of e x c e s s i v e l y f l o o d i n g the s o i l .  Any excess water t h a t may have t r i c k l e d down through cracks  i n the f r o z e n s o i l surface was drained through the lower f l u s h i n g tube. an "ice-sheet approximately  Once  3 t o 4 cm. t h i c k h a d b e e n formed so t h a t i t j u s t - ,  27 , covered the' p r o t r u d i n g s o i l atmosphere sampling tubes, the rubber f l u s h i n g tubes were c l o s e d t i g h t l y with screw clamps o r corks. I f the experiment  i n v o l v e d f l u s h i n g the s o i l with e i t h e r oxygen,  n i t r o g e n ' o r carbon d i o x i d e , the lower f l u s h i n g tube .'was'connected''.to a :  c y l i n d e r o f the compressed gas, and a steady stream, a t approximately 200 ml./min. was permitted t o flow through the bottom o f the s o i l and out • through the upper flushing, tube..  When the-experiment  i n v o l v e d passing a i r  through the s o i l ,a c i r c u l a t i n g pump was used.  2 i 6 . S O I L ATMOSPHERE-SAMPLING AND ANALYSIS  To analyze the composition of the s o i l atmosphere under the i c e - s h e e t , hot water was at f i r s t poured on the surface o f the i c e j u s t above the sampling tube.  Enough water was poured to melt out a small hollow so as t o  expose the s i l i c o n e rubber septum.  Two 5 c.c. gas samples were, then w i t h r  drawn.through the silicone•septum-using a 5 c.c. c a p a c i t y g a s ' t i g h t "B-D Hypak D i s c a r d i t " syringe.with a B-D Yale 26 gauge, -jr-inch l o n g disposable needle.  The.samples'were analyzed i n a Shimadzu type GO - 2B gas-chromatpgraph.  The chromatograph was equipped with two-copper columns w i t h an i n s i d e of  5 nim..  One column,. 3 m. long, was packed with, s i l i c a g e l , which separates  carbon d i o x i d e from oxygen and n i t r o g e n . was  diameter  The second column, 1.6 m. l o n g  packed w i t h Linde molecular sieve 5 A, which i r r e v e r s i b l y absorbs  d i o x i d e , but separates oxygen from n i t r o g e n .  carbon  Helium was used as the c a r r i e r  gas at a flow r a t e of 75 ml./min. and an i n l e t pressure of 1.2 kg./cm . A tungsten f i l a m e n t d e t e c t o r was used, and the data recorded on a Shimadzu  28 recorder with a 2.0 m i l l i v o l t range. . The- gas-chrqmatograph  was,,calibrated  p e r i o d i c a l l y , with, known, yolumes of oxygen, n i t r o g e n .and, c a r b o n - d i o x i d e ,  2.7.  At  INJURY CRITERIA  the end of t h e i r , period i n the ~3.0°C room-, the plants'-,. s t i l l .  mounted in" the boxes, were moved outdoors. ,Gool winter temperatures permitted a gradual thawing ,of the i c e - s h e e t . . Once t h e - i c e had melted,- the pots were removed from the boxes with temperature c o n t r o l and,moved to a greenhouse. Extent of damage was assessed by sectioning,and v i t a l - s t a i n i n g , as-well as '. by,measuring recovery, r a t e s . F o l l o w i n g an-, 8-day recovery period i n the greenhouse,  sections  were made of l i v e t i s s u e by mounting approximately... a 2 cm.; long, p o r t i o n of ..the-, crown and the upper part of the tap-root i n b a l s a wood.- -..The wood was then clamped i n - a s l i d i n g microtome and:-by-using s t a i n l e s s - s t e e l r a z o r blades complete cross s e c t i o n s - 4 0 m i c r o n s ; t h i c k , of the t i s s u e immediately .beneath the. crown, could be made.-A • s l i g h t l y - m o d i f i e d v i t a l , s t a i n i n g .technique., described by Luyet,,(86;),.;was used.  The s e c t i o n s were.placed on a microscope,  s l i d e ,and a.few drops o f a 0:.slightly red  alkaline,'aqueous s o l u t i o n , of n e u t r a l  were deposited on-the: sections;and l e f t there, f o r 2 minutes;  .then the;.,  neutral- red s o l u t i o n was, b l o t t e d o f f - and,:replaced .by a.,drop, of 0.25$ '. aqueous potassium,hydroxide, which was, immediately:removed.-with  a blotter.--  A f e w . d r o p s , o f water- were,:;deposited-on the section,, and a coyer g l a s s was. then placed over i t .  The .tissue was immediately examined under a .microscope.,  T h i s s t a i n i n g technique, resulted,in.,the - l i v i n g - c e l l s , t a k i n g on a b r i g h t c e r i s e red  c o l o r , while the dead c e l l s were an intense orange-yellow.  29 Recovery r a t e s were measured by t a k i n g oven-dry weights of the a e r i a l p o r t i o n s o f the p l a n t s , u s u a l l y f o l l o w i n g a 14-day period i n the greenhouse-..  2.8.  . Since the composition  ROOT MEASUREMENTS  of -the s o i l - atmosphere under an ice-sheet  i s dependent to a c e r t a i n extent on the r e l a t i v e weights o f s o i l and roots i n each pot, f o l l o w i n g -the recovery•period  i n the greenhouse, .the roots of  each plant were then oven d r i e d a t 90.0°C f o r 24 hours and weighed.  INSULATED  BOX  WITH 4  POT  TEMPERATURE  CAPACITY  FIG  2.1  CONTROL  SINGLE  POT  MOUNTED  IN  BOX  WITH  TEMPERATURE  CONTROL  ARRANGEMENT  OF  BOXES ON  WITH  TEMPERATURE  CONTROL  DEXIO  -THAWING 6 0 W.  LIO with 2  LIGHT  COVERING  CUT  AWAY  CONTINUOUS 3  BULBS 2 BOXES  VIEW  ICE S H E E T  ADJACENT  COVERING  BOXES  Vol  FIG 2 . 3  33  1  1 •  E  m  ri i  1 —  >  Fig.  2.4  Painted p l a s t i c pot with top sealed, mounted i n 00^ f i l l e d p l e x i g l a s s chamber. Gases i n the pot were withdrawn through a s i l i c o n e septum mounted i n the upper end of the tube protruding through the l i d .  Fig.  2.5  Rotary switch connecting 11 thermocouples mounted i n d i f f e r e n t l o c a t i o n s . Darker cords seen on lower r i g h t connect Bouyoucos moisture blocks i n the s o i l beneath the i c e - s h e e t .  34 Arrangement o f boxes with temperature c o n t r o l on the Dexion frame i n the c o l d room. A continuous i c e sheet covering two adjacent boxes can be seen on the surface of the upper boxes. A c y l i n d e r of CO^ was used i n s o i l f l u s h i n g experiment.  Fig. 2.7 Manometers were attached to f l u s h i n g tubes to detect any pressure change that may have taken place i n the s o i l under an i c e - s h e e t .  35  Fig.  2.8  Thawing-lid, i n s u l a t e d and equipped w i t h two 60 watt l i g h t b u l b s , which provided enough heat t o r a i s e the temperature above 8 pots t o 15°C. Thermocouple w i r e s l e a d i n t o the cover f o r temperature measurement. Temperature under the cover was c o n t r o l l e d by a r h e o s t a t connected t o the l i g h t b u l b s . An i c e - s h e e t can be seen i n the lower l e f t corner.  36  3.  EXPERIMENTATION  Using the m a t e r i a l s and methods described, a s e r i e s of experiments were performed alfalfa  to determine the nature of i c e - s h e e t damage to overwintering  a t temperatures which per se are not i n j u r i o u s to the p l a n t s .  D e s c r i p t i o n s of the experiments are given i n the sequence i n which they were c a r r i e d out.  3.1.  EXPERIMENT I  The e f f e c t of an ice-sheet cover on the composition of the s o i l atmosphere and on -the recovery of the p l a n t s which had been covered by i c e .  Eight  'Vernal' a l f a l f a  p l a n t s were•transplanted from the f i e l d  painted p l a s t i c pots on October 6, 1965.  into  Since the p l a n t s were not completely  winter hardened t h i s e a r l y i n the f a l l , they were kept f o r 2 weeks i n the  near 0°C room.  They were then moved i n t o the -3.0°C room and 4 p l a n t s were  covered i n an ice-sheet;  the other 4 were not i c e covered.  The p l a n t s were  kept i n the f r e e z i n g room f o r 38 days, and f o l l o w i n g an a n a l y s i s of the s o i l atmosphere, were moved to the greenhouse.  3.2.  3.2-1.  EXPERIMENT I I  The e f f e c t on p l a n t s of f l u s h i n g an i c e covered s o i l with CO,,.  On November 10, 1965, sixteen 'Vernal' a l f a l f a p l a n t s were t r a n s planted from the f i e l d i n t o p l a s t i c pots, and moved i n t o the n e a r - f r e e z i n g temperature  (0°C + 1.0°C) room f o r 1 week.  They were then t r a n s f e r r e d to  the -3.0°C room, and f o l l o w i n g the formation of an ice-sheet on the surface, the f o l l o w i n g treatments were a p p l i e d over a 62-day periods (i)  covered by an ice-sheet, s o i l f l u s h e d with 0^, 4 pots  (ii)  covered by an ice-sheet, s o i l f l u s h e d with CO , 4 pots  (iii)  no i c e - s h e e t , s o i l flushed with 0^, 4 pots  (iv)  no i c e - s h e e t , s o i l f l u s h e d with 00^, 4 pots  The s o i l was f l u s h e d once a week, by passing a stream of gas through the f l u s h i n g tubes f o r approximately 5 minutes, a f t e r which the tubes were closed t i g h t l y u n t i l the next f l u s h i n g p e r i o d . T h i s experiment, with-a few m o d i f i c a t i o n s , was repeated, u s i n g greenhouse grown 'Dupuits' a l f a l f a and a i r - t i g h t metal pots.  The p l a n t s  were seeded on November 5, 1965 and were t r a n s f e r r e d f o r hardening to a c o l d frame out-of-doors on February 24, 1966, where they were kept f o r a 2 week p e r i o d .  E i g h t p l a n t s were then moved to the near 0°C room f o r 1 week;  near 0 C room  0  They were then moved i n t o the -3«0 C room and 4 p l a n t s were  covered i n an i c e - s h e e t ;  the other 4 were not i c e covered.  The p l a n t s were  kept i n the f r e e z i n g room f o r 38 days, and f o l l o w i n g an a n a l y s i s o f the s o i l atmosphere, were moved t o the greenhouse.  3.2.  3.2-1.  EXPERIMENT I I  The e f f e c t on p l a n t s o f f l u s h i n g an i c e covered s o i l w i t h CO^. On November 10, 1965, s i x t e e n 'Vernal' a l f a l f a p l a n t s were t r a n s -  planted from the f i e l d i n t o p l a s t i c pots, and moved i n t o the n e a r - f r e e z i n g temperature (0°C _+ 1.0°C) room f o r 1 week.  They were then t r a n s f e r r e d t o  the -3.0°C room, and f o l l o w i n g the f o r m a t i o n o f an ice-sheet on the s u r f a c e , the f o l l o w i n g treatments were a p p l i e d over a 62-day periods (i)  covered by an i c e - s h e e t , s o i l f l u s h e d w i t h 0^, 4 pots  (ii)  covered by an i c e - s h e e t , s o i l f l u s h e d w i t h CO^, 4 pots  (iii)  no i c e - s h e e t , s o i l f l u s h e d w i t h 0^, 4 pots  (iv)  no i c e - s h e e t , s o i l f l u s h e d w i t h COg, 4 pots  The s o i l was f l u s h e d once a week, by p a s s i n g a stream o f gas through the f l u s h i n g tubes f o r approximately 5 minutes, a f t e r which the tubes were c l o s e d t i g h t l y u n t i l the next f l u s h i n g p e r i o d . T h i s experiment, w i t h a few m o d i f i c a t i o n s , was repeated, u s i n g greenhouse grown 'Dupuits' a l f a l f a and a i r - t i g h t metal pots.  The p l a n t s  were seeded on November 5, 1965 and were t r a n s f e r r e d f o r hardening t o a c o l d frame out-of-doors on February 24, 1966, where they were kept f o r a 2 week p e r i o d .  E i g h t p l a n t s were then moved t o the near 0°C room f o r 1 week;  38 f o l l o w i n g t h i s they were t r a n s f e r r e d t o the f r e e z i n g room, where an i c e - s h e e t was formed which covered the 8 p o t s .  The f o l l o w i n g treatments were then a p p l i e d  over a 3-week periods (i)  covered hy an i c e - s h e e t , s o i l f l u s h e d w i t h a continuous stream o f a i r , 4 pots  (ii)  covered hy an i c e - s h e e t , s o i l f l u s h e d w i t h CCV, approximately 4 hours each day, 4 pots  3.2.2.  Do h i g h CO^ l e v e l s have an a p p r e c i a b l e e f f e c t on the s o i l pH? To determine whether o r not the h i g h CC^ l e v e l s , such as were created  i n the experiments j u s t d e s c r i b e d , had any i n f l u e n c e on the s o i l pH, 4 metal pots were f i l l e d w i t h the f i e l d s o i l and were c l o s e d w i t h a i r - t i g h t  lids.  Carbon d i o x i d e was passed i n t e r m i t t e n t l y through the pots f o r 2 weeks. I n between the f l u s h i n g p e r i o d s a l l connecting tubes were c l o s e d t i g h t l y t o m a i n t a i n a h i g h CO^ l e v e l w i t h i n the p o t . The s o i l moisture content, on an oven d r y b a s i s , was 34$. A t the end o f the 2 week p e r i o d e l e c t r o d e s from a pH meter were i n s e r t e d i n t o the s o i l and the pH was measured.  I n cases where  the f l o w o f c u r r e n t was e r r a t i c , a d d i t i o n a l water was s q u i r t e d around the electrodes.  S o i l samples from the 4 pots were a l s o placed i n beakers, t o  which approximately an equal volume o f water was added, forming a s l u s h .  The  pH o f the s l u s h was found t o be much the same as the s o i l which ranged from 5.6 t o 6.2.  39  3.3.  3.3.1.  EXPERIMENT I I I  The r o l e s o i l moisture plays i n the a b i l i t y of a l f a l f a  to survive an  ice-sheet cover.  3.3.2.  E s t i m a t i o n of the amount of CO^ accumulated s o i l microorganism  3.3.3.  and 0 ^ depleted due to  a c t i v i t y under an i c e - s h e e t .  Comparison of the composition of the s o i l atmosphere under an i c e - s h e e t when the f l u s h i n g tubes have been kept open, with that of a s o i l under an i c e - c o v e r , when the tubes have been kept closed f o r the same length of time;  to compare plant responses to these two  On november 2 9 , 1 9 6 5 , fourteen 'Vernal' a l f a l f a  conditions.  p l a n t s were t r a n s -  planted from the f i e l d i n t o metal pots and kept f o r 7 days i n the 0°C room. Two  pots were f i l l e d with f i e l d s o i l taken from between the rows of  used i n the experiments.  alfalfa  Once an ice-sheet had been formed on the s u r f a c e ,  the f o l l o w i n g treatments were a p p l i e d over a 5 0 day periods  (i)  covered by an i c e - s h e e t , s o i l c o n t a i n i n g no p l a n t s , 2 pots  (ii)  covered by an i c e - s h e e t , s o i l with p l a n t s water-saturated, 4  (iii)  pots  covered by an i c e - s h e e t , s o i l with p l a n t s , s o i l moisture a t approximately 3 0 $ of dry weight and aerated by l e a v i n g the :  f l u s h i n g tubes open, 4 pots. (iv)  covered by an i c e - s h e e t , s o i l with p l a n t s , s o i l moisture at approximately 3 0 $ of dry weight, manometers attached to lower  40  f l u s h i n g tubes, i . e . system was gas t i g h t , 4 pots (v)  no ice-sheet cover, s o i l with p l a n t s , s o i l moisture a t approximately 30$ of dry weight, 2 pots In t h i s experiment s o i l , moisture was measured u s i n g c a l i b r a t e d  Bouyoucos s o i l moisture b l o c k s .  3.4.  3.4.1.  EXPERIMENT IV  The extent to which a freeze-thaw-freeze  c y c l e , a s s o c i a t e d with an i c e -  sheet, i s damaging to a l f a l f a .  3.4.2.  Does stubble, p r o t r u d i n g through an i c e - s h e e t , serve as a passage f o r gas d i f f u s i o n to the root system, and i n t h i s way be of some p r o t e c t i o n against ice-sheet damage?  On January 19, 1966, s i x t e e n 'Dupuits'  alfalfa  p l a n t s were t r a n s -  planted i n t o metal pots, and f o l l o w i n g the usual 7-day period i n the 0°C room, they were moved i n t o the -3.0°C room.  An ice-sheet was formed on the  surface of 12 p l a n t s , while the remaining 4 were not covered by i c e .  The  f o l l o w i n g treatments were a p p l i e d over a period of 40 days:  (i)  no i c e - s h e e t , subjected to a 20-day freeze period followed by a 10-day thaw then a 10-day freeze period again, 4 pots  (ii)  subjected to a 20-day ice-sheet cover followed by a 10-day thaw then a 10-day ice-sheet cover again, 4 pots  (iii)  subjected to a continuous 40 day i c e - c o v e r with stubble through the i c e - s h e e t , 4 pots  protruding  41  (iv)  e n t i r e l y encased i n i c e f o r the f u l l 4 0 - d a y p e r i o d , 4 pots  3.5.  EXPERIMENT V  Does a high s o i l moisture content  i n the winter r e s u l t i n an  increased  hydration l e v e l i n the t i s s u e which i n t u r n increases the p l a n t s ' s u s c e p t i b i l i t y to f r e e z i n g ?  Three p o l y e t h y l e n e - l i n e d crates ( P i g . 3«l) were f i l l e d with f i e l d and on February 2 2 , 1 9 6 6 , twelve f i e l d grown 'Ladak' a l f a l f a p l a n t s were transplanted i n t o each of the c r a t e s . 0°C room.  The  s o i l i n one  The  crates were then moved i n t o  of the c r a t e s was  saturated with water, i n the  other wetted to approximately i t s f i e l d c a p a c i t y , while i n the t h i r d , p r e v i o u s l y room-dried s o i l was i n the s o i l s at the 4 5 . 7 $  3  l e f t i n i t s dry s t a t e .  The  d i f f e r e n t moisture l e v e l s , namely  of the dry weight, f o r  12  the  the  p l a n t s were l e f t  15.8$,  32.1$,  and  days, at the end of which the p l a n t s were  removed. S o i l from the r o o t s of 8 plants from each of the c r a t e s was washed o f f u s i n g a minimum of water.  Fibrous roots were then cut o f f , l e a v i n g the  tap root, a few bigger branch r o o t s , and the crown.  The  p l a n t s were then  b l o t t e d dry on the surface, q u i c k l y weighed, and placed i n an oven set at 9 0 ° C f o r 2 4 hours. of the t i s s u e was  A f t e r d r y i n g and weighing again the moisture percentage determined.  S o i l adhering to the remaining 4 p l a n t s from each of the c r a t e s was  shaken o f f and the p l a n t s were placed i n p l a s t i c bags f o r a period of  4 hours i n the - 3 . 0 ° C room.  Following t h i s period the bags were moved to a  soil,  42 -10oO°C room, where they were l e f t f o r 48 hours.  The plants were then removed  and immediately sectioned i n the region j u s t below the crown, using a s l i d i n g microtome.  The  sections were stained with n e u t r a l red and  hydroxide and examined under a microscope f o r damage due remaining part of each root was  potassium  to f r e e z i n g .  cut i n t o approximately 1 cm.  The  long sections  and dropped i n t o 50 ml. Erlenmeyer f l a s k s containing 10 ml. tap water. technique described by Siniinowitch et a l . ( l l 8 ) was extent to which the t i s s u e was According  A  followed to determine the  damaged as a r e s u l t of the f r e e z i n g treatment.  to Siminowitch et a l . ( l l 8 ) the determination  of the  concentration  of amino acids i n the water i n which the plant t i s s u e s are leached a f t e r f r e e z i n g , can be used as a q u a n t i t a t i v e method f o r the estimation of the i n j u r y sustained i n the f r e e z i n g .  Fig.  5.1  Polyethylene l i n e d crates used i n Experiment VI, to determine the e f f e c t of s o i l moisture on the hydration l e v e l of t i s s u e of overwintering p l a n t s .  43 3.6.  EXPERIMENT VI  Is CO^ accumulation o r 0 ^ d e p l e t i o n o r both, the causal f a c t o r i n ice-sheet i n j u r y ?  For t h i s experiment months i n the greenhouse.  a clone of 'Vernal' a l f a l f a was grown f o r 2?  F o l l o w i n g a 2-week hardening p e r i o d i n a c o l d frame  and 1-week i n the 0°C room, 8 p l a n t s were covered by an i c e - s h e e t f o r 21 days. The f o l l o w i n g treatments were then a p p l i e d :  (i)  s o i l f l u s h e d almost continuously with C^, 2 pots  (ii)  s o i l f l u s h e d almost continuously with CO - f r e e a i r , 2 pots  (iii)  s o i l f l u s h e d with CO^ f o r approximately 4 hours each day, 2 pots  (iv)  s o i l f l u s h e d almost continuously with TS , 2 pots  F l u s h i n g the s o i l with CO^-free a i r was achieved by scrubbing the pumped a i r through 3 f l a s k s of 20$ NaOH and f i n a l l y  passing i t over " A s c a r i t e "  packed i n t o a g l a s s tube 30 cm. long. To c o l l e c t the CO^ evolved by the s o i l , while f l u s h i n g with 0^, N , and a i r , the outflowing gas was f i r s t d r i e d by passing i t through cone. S^SO^ and a tube 30 cm. long of anhydrous CuSO^, and then through a 20 ml. " Q u i c k f i t " separatory funnel packed with " A s c a r i t e " ( F i g . 3.2). To determine the amount of 00^ evolved the separatory funnels were removed and weighed.  44  Fig. 3.2 F l a s k s of cone. H2SO4 and l o n g white tubes of anhydrous CUSO4 were used to dry the outflowing gas. CO2 was then c o l l e c t e d on " A s c a r i t e " packed i n t o small separatory funnels seen above. Small tubes a l s o packed with " A s c a r i t e " were attached to the o u t l e t end of the separatory funnels to ensure that no atmospheric C 0 would be absorbed. o  3.7.  EKPERIMENT V I I  The reaction of different species and varieties of several species of forage p l a n t s , to withstand a prolonged period of i c e encasement.  A technique of encasing an e n t i r e potted plant o r a t u r f plug i n i c e , has been used by S j o s e t h (ll9) and Beard (6) i n studies to determine v a r i e t a l d i f f e r e n c e s i n the a b i l i t y of d i f f e r e n t species to withstand i c e encasement.  The technique involved p l a c i n g the pots o r t u r f plugs i n the  45 center of a volume of water, and p e r m i t t i n g them to freeze i n a r e f r i g e r a t e d room.  Under such c o n d i t i o n s the f r e e z i n g plane progressed  r e s u l t i n g i n a build-up of h y d r o s t a t i c pressure  i n the center of the  Under n a t u r a l c o n d i t i o n s the f r e e z i n g plane progresses v e r t i c a l l y down, and normally no h y d r o s t a t i c pressure the f r e e z i n g plane.  Thus, h i g h l y unnatural  from a l l d i r e c t i o n s ,  only i n one  soil.  direction—  i s formed i n f r o n t of  conditions are created by f r e e z i n g  t e s t p l a n t s i n the manner described by Sjoseth ( l l 9 ) and Beard ( 6 ) . The  species and v a r i e t i e s used i n t h i s experiment were grown i n  p l a s t i c pots, i n a greenhouse with supplementary l i g h t i n g . v a r i e t y was at  planted every second day, beginning  Each species  on November 1, 1965,  and  so that,  the time of f r e e z i n g a l l p l a n t s would be of a comparable p h y s i o l o g i c  age.  Handling of the d i f f e r e n t p l a n t s i n d i v i d u a l l y , however, l a t e r proved to be i m p r a c t i c a l and on February 24, 1966  a l l the p l a n t s were c l i p p e d down to  approximately a 4 cm. height and were t r a n s f e r r e d to a c o l d frame. time the p l a n t s had been thinned out to a s i n g l e plant per pot. 1966  By that  On March 14,  a l l the species and v a r i e t i e s were moved to the near 0°C room.  A  technique devised by Taber ( l 2 6 ) , i n h i s c l a s s i c work on s o i l heaving, used to f r e e z e the potted s o i l from the surface down. the use of a h e a v i l y i n s u l a t e d box f i l l e d with sand.  The  technique involved  Eight pots of a s i n g l e  v a r i e t y or species were embedded i n the sand, to a depth, so that the of the s o i l i n the pot was The  sand box was  48 hours.  The  surface  even with the surface of the sand. ( F i g . 3»3).  then wheeled i n t o the -3.0°C room, where i t remained f o r  f r e e z i n g progressing from the surface down could be  by a s e r i e s of thermocouples mounted at 2.5 was  was  d r i v e n i n t o the sand.  cm.  i n t e r v a l s on a stake, which  A f t e r 48 hours the s o i l was  and the 8 pots were removed.  followed  completely  frozen,  46  Fig.  5.5  H e a v i l y i n s u l a t e d sand box used t o f r e e z e the s o i l i n pots from the surface down. Temperatures a t d i f f e r e n t depths i n the sand were measured w i t h thermocouples mounted on a stake.  The sand box was then wheeled out o f the f r e e z i n g room i n t o a heated room, so as to warm the sand to about 5.0°C p r i o r t o embedding the next batch of 8 p l a n t s . day.  I n t h i s manner 8 pots were f r o z e n approximately every second  F o l l o w i n g removal from the sand, 4 pots were stored i n the -3.0°C room  without f u r t h e r treatment, w h i l e 4 were placed i n wax paper cartons 18 cm. h i g h and an i n t e r n a l diameter of 13 cm. the  Chipped i c e was packed t i g h t l y around  pots i n the wax c a r t o n s , and they were then f i l l e d to c a p a c i t y w i t h a  f r e e z i n g water m i x t u r e . The water surrounding the pot f r o z e r a p i d l y , so t h a t extensive thawing of the s o i l was improbable. A f t e r b e i n g stored i n the f r e e z i n g room f o r 2 months, the 8 p l a n t s of each v a r i e t y o r species were moved t o the greenhouse f o r a 2-week recovery p e r i o d .  So as not t o f l o o d the 4 p l a n t s which had been encased  47 i n i c e , the wax paper was t o r n o f f immediately f o l l o w i n g removal from the -3»0°C room.  A t the end o f a 2-week p e r i o d i n the greenhouse, the p l a n t  tops were c u t , oven d r i e d and weighed.  The weight o f the tops produced d u r i n g  the recovery p e r i o d i n the greenhouse was used as a c r i t e r i o n f o r a s s e s s i n g damage s u f f e r e d hy the p l a n t s i n the f r e e z i n g room.  The s o i l o f the 4 potted p l a n t s on the r i g h t i s f r o z e n w h i l e the 4 pots on the l e f t a r e e n t i r e l y surrounded by i c e . A l l 8 pots were s t o r e d a t -3.0°C f o r 2 months.  48  The the Ice the  wax c a r t o n was t o r n o f f immediately a f t e r the pots had "been removed from -3.0 C room, so that water from the m e l t i n g i c e would not f l o o d the p l a n t s . encased pot immediately a f t e r the c a r t o n had been removed can be seen i n center.  49  4.  OBSERVATIONS AND RESULTS  4.1.  EXPERIMENT I  The e f f e c t . o f an i c e - s h e e t on the composition of the s o i l atmosphere and on the recovery of the p l a n t s which had been covered by i c e .  The 4 p l a n t s subjected to an i c e - s h e e t cover of 38 days s u r v i v e d , but t h e i r recovery i n the greenhouse was slower than the recovery of the 4 p l a n t s which had no i c e - c o v e r ( F i g . 4«l).  The composition of the s o i l  atmosphere a t the end of the 38 day period i s given i n Table 4.1.  50  TABLE 4.1. COMPOSITION OP THE SOIL ATMOSPHERE WITH AND WITHOUT AN ICE-COVER AFTER 38 DAYS AT -3.0°C Composition o f s o i l atm. (per cent) Treatment Eot no. Ice-sheet  No ice-sheet  0„  C0  1 2 3 4  7.0 4.0 6.0 6.0  2.6 5.2 3.6 4.0  1 2  22.0 22.0 22.0 22.0  3 4  1 M  I  p.  *  0.02 0.02 0.02 0.02  i f  Roots 1.80 2.05 1.95 1.90  Dry wt. s o i l (gm.)  Tops  Tot.  4086 3831 4426 4226  3.30  -  --  Tot.  -  6.35  -  \  ism  - f i U VERNAL ALFALFA  KEPT 38 DAYS AT -30°C NO ICE SHEET RECOVERY  o  Dry wt. p l a n t f o l l o w i n g 20 day recovery (gm.)  FOLLOWING  2 0 DAYS  IN  GREEIHOUSE  VERNAL ALFALFA KEPT 38 DAYS AT -30°C COVERED BY AN ICE SHEET RECOVERY  FOLLOWING  2 0 DAYS  IN  GREENHOUSE  Figo 4.1  Growth o f the 4 p l a n t s a f t e r an ice-sheet cover o f 38 days, was slower than the growth o f the p l a n t s t h a t were not covered hy i c e .  * S o i l and r o o t s from the pots, t h a t had no i c e - c o v e r were not weighed, s i n c e the composition of the atmosphere i n each o f these pots was independent o f the two weights.  51 In the ice-sheet covered pots, CO^ to as high as 5.2$.  This highest CO^  accumulated i n the s o i l atmosphere  accumulation was  a t t a i n e d i n a pot with  the smallest quantity of s o i l and the l a r g e s t root system of the 4 pots used i n the experiment.  The highest CO^  accumulation r e s u l t e d i n the greatest  0^  d e p l e t i o n , or the ice-sheet i t s e l f or even a combination of these f a c t o r s appeared to be damaging to the p l a n t s , since the dry weight of the shoots, f o l l o w i n g a 20-day recovery period, of the p l a n t s not covered by an  ice-sheet  was  composition  almost double that of the plants that were covered by i c e .  of the s o i l atmosphere i n the pots that had no ice-cover was  The  very s i m i l a r to  the composition of a i r .  4.2.  4.2.1.  EXPERIMENT I I  The e f f e c t on plants of f l u s h i n g an i c e covered s o i l with  CO^.  Of the 16 p l a n t s used i n t h i s experiment, a l l recovered v i g o r o u s l y f o l l o w i n g t h e i r 62-day period i n the -3.0°C room, except the plants that were covered by i c e and had CO^ T h i s r e s u l t shows that high l e v e l s of CO^  f l u s h e d through the s o i l ( P i g . 4.2).  i n the s o i l associated with an  ice-sheet are extremely damaging to plants.by i c e but had  4  The 4 p l a n t s which were covered  0^ f l u s h e d through the s o i l , grew as v i g o r o u s l y as d i d the  plants which were not covered by an i c e - s h e e t .  An ice-sheet per se, t h e r e f o r e ,  i s not damaging to a l f a l f a . Some d i s r u p t i o n of the t i s s u e was were covered by i c e and had 0  found i n sections of plants that  f l u s h e d through the s o i l ( F i g . 4.3').  This  52 s l i g h t d i s r u p t i o n , mainly i n the phloem and p e r i c y c l e , a p p a r e n t l y d i d not h i n d e r the p l a n t s ' recovery i n the greenhouse.  No damage could be observed  i n s e c t i o n s of the p l a n t s t h a t were not covered by i c e f o r the 62-day p e r i o d (Fig.  4.4).  V i t a l s t a i n i n g of the s e c t i o n s revealed dead t i s s u e i n p l a n t s  which had been covered by i c e and which had CO^ f l u s h e d through the s o i l . Death a p p a r e n t l y was not due to mechanical d i s r u p t i o n ( F i g . 4.5).  On the  o t h e r hand the t i s s u e of both the i c e encased p l a n t s t h a t had 0^ f l u s h e d through the s o i l as w e l l as the p l a n t s t h a t were not covered by i c e , s t a i n e d a c e r i s e - r e d , which i s i n d i c a t i v e of the presence of l i v e c e l l s ( F i g . 4.6,  ^g.  4.2  Ice covered s o i l through which CO2 was f l u s h e d r e s u l t e d i n the death of the p l a n t s . When Op was f l u s h e d through the s o i l , the p l a n t s recovered as v i g o r o u s l y as those which had no i c e - c o v e r .  4.7).  53  Fig.  4.5  C r o s s - s e c t i o n o f the r e g i o n j u s t below the crown of an a l f a l f a p l a n t encased i n i c e f o r 62 days. The s o i l was f l u s h e d w i t h 0 - Some mechanical damage t o the phloem and p e r i c y c l e can be seen, (p) p e r i c y c l e , (ph) phloem, (c) cambium, (x) xylem r a y 2  Fig.  4.4  C r o s s - s e c t i o n o f the r e g i o n j u s t below the crown o f an a l f a l f a p l a n t kept f o r 62 days a t - 3 . 0 ° C without an. i c e - c o v e r . Bb mechanical damage i s v i s i b l e .  54  V i t a l staining' of a s e c t i o n taken j u s t below the crown of a plant kept f o r 62 days covered by an ice-sheet. Carbon dioxide was flushed p e r i o d i c a l l y through the s o i l . The yellow s t a i n i n g t i s s u e i s dead. No l i v e , red s t a i n i n g c e l l s could be found, (y) yellow s t a i n i n g t i s s u e  V i t a l s t a i n i n g of a s e c t i o n taken j u s t below the crown of a plant kept f o r 62 days covered by i c e . The s o i l was f l u s h e d p e r i o d i c a l l y with 0^. The deep red s t a i n i n g t i s s u e i s a l i v e , ( r ) red s t a i n i n g t i s s u e  Fig.  4.7  V i t a l staining- of a s e c t i o n taken j u s t below the crown of a plant kept f o r 62 days at -3.0°C without an i c e - c o v e r . Most of the xylem ray c e l l s are a l i v e and s t a i n r e d .  Fig.  4.8  The s o i l i n the 4 pots was covered by an i c e - s h e e t . The s o i l of the 2 pots on the l e f t was aerated by a continuous stream of a i r , while the 2 pots on the r i g h t were p e r i o d i c a l l y f l u s h e d with CO-.  56  Figp 4.9 V i t a l s t a i n i n g of a s e c t i o n j u s t below the crown of a p l a n t kept f o r 21 days covered by an i c e - s h e e t and the s o i l f l u s h e d c o n t i n u o u s l y w i t h a i r .  Fig.  4.10  V i t a l s t a i n i n g of a s e c t i o n j u s t below the crown of a p l a n t kept f o r 21 days covered by an i c e - s h e e t and the s o i l f l u s h e d i n t e r m i t t e n t l y w i t h CO,,. Sections were made a f t e r an 8-day recovery p e r i o d i n the greenhouse.  57 Passing e i t h e r 0^ or CO^ through the s o i l f o r approximately 5 minutes, once a week was not s u f f i c i e n t to f o r c e a l l the other gases out of the s o i l atmosphere.  The s o i l atmosphere was analyzed  62-day period, 1 week a f t e r the l a s t f l u s h i n g treatment„  at the end of the The CO,, i n the i c e -  sheet covered s o i l , f l u s h e d with CO^ gas was found t o be 30$ o f the s o i l atmosphere.  Carbon dioxide accumulated quite r a p i d l y i n the 0^ f l u s h e d  and a week a f t e r f l u s h i n g , the composition sheet was:  CO^ - 2$, CV, - 15$,  soil  o f the s o i l atmosphere under the i c e -  and the r e s t presumably n i t r o g e n . *  With a s l i g h t m o d i f i c a t i o n i n procedure, CO,, ws)S again f l u s h e d through the s o i l of i c e - s h e e t covered pots, i n which greenhouse grown p l a n t s were rooted.  These p l a n t s were younger than those used i n the experiment j u s t  described.  The 4 p l a n t s covered by i c e , which had CO,, f l u s h e d i n t e r m i t t e n t l y  through the s o i l f o r 21 days, were k i l l e d and d i d not recover i n the greenhouse ( P i g . 4.8). On the other hand the 4 p l a n t s that were a l s o i c e covered, but had a i r continuously passed through the s o i l , grew v i g o r o u s l y i n the greenhouse ( F i g . 4.8)..  Following an 8-day recovery p e r i o d , 4 plants were  sectioned and s t a i n e d with a v i t a l s t a i n . seen.  No mechanical d i s r u p t i o n could be  V i t a l s t a i n i n g revealed that the t i s s u e of the p l a n t s exposed t o high  CO,, l e v e l s under the ice-sheet was dead, and that the t i s s u e o f the p l a n t s rooted i n aerated s o i l stained red and was a l i v e ( F i g .  4 . 9 , 4.10).  Again, the r e s u l t s showed that the composition  o f the s o i l atmosphere  was the c r i t i c a l f a c t o r i n the s u r v i v a l o f i c e covered p l a n t s , and that the ice-sheet per se was not i n j u r i o u s t o overwintering  alfalfa.  •Nitrogen l e v e l s i n the" s o i l atmosphere could hot be determined since a t the s e n s i t i v i t y "which O2 and CO2 was analyzed, the N2 peak was bigger than the f u l l s c a l e on the recorder.  58  4.2.2.  Do high GO^ l e v e l s have an appreciable  The  f i e l d s o i l used i n the experiments appears to have been  s u f f i c i e n t l y b u f f e r e d to prevent a lowering i n the s o i l water. w i t h CO  f o r 2 weeks, no change i n the s o i l pH could be detected.  EXPERIMENT I I I  The r o l e s o i l moisture plays i n the a b i l i t y ice-sheet  4.3.2.  o f i t s pH by s o l u t i o n of CO^  A f t e r i n t e r m i t t e n t l y f l u s h i n g a wet s o i l kept a t 1.0°C  4.3.  4.3.1.  e f f e c t on the s o i l pH?  of a l f a l f a  t o s u r v i v e an  cover.  E s t i m a t i o n of the amount o f 00^ accumulated and 0^ depleted due to s o i l microorganism a c t i v i t y under an i c e - s h e e t .  4.3.3.  Comparison o f the composition of the s o i l atmosphere under an ice-sheet when the f l u s h i n g tubes have been kept open, w i t h that of a s o i l under an i c e - c o v e r , when the tubes have been kept closed f o r the same length of time; The  t o compare the plant responses t o these two conditions.'  4 i c e covered-plants,  rooted i n a , s o i l ' w i t h a"moisture l e v e l  approximately a t f i e l d c a p a c i t y and-where the. f l u s h i n g tubes were kept c l o s e d , were killed-,  excepting  1 plant which b a r e l y survived t h i s treatment and grew  weakly i n the greenhouse ( P i g . 4 . 1 l ) .  During the 50-day period under, gas  t i g h t c o n d i t i o n s , CO^ accumulated i n one o f the.pots to as high as 10$, while 0^ was depleted  to 3.7$ of the s o i l atmosphere.  0^ l e v e l s , recorded  Even lower 00^, and higher  in- the other pots, proved to be damaging to the p l a n t s . .  Furthermore, on s e c t i o n i n g one of the p l a n t s a f t e r a 10-day recovery no mechanical damage could be detected.  period,  V i t a l s t a i n i n g , however, revealed  that  59 only a few of the c e l l s were a l i v e , and that most of the- t i s s u e was-dead ( F i g . 4.15)• Damage, t h e r e f o r e , appeared to be p h y s i o l o g i c a l r a t h e r than mechanical.. The 4 p l a n t s that were covered by i c e and where the f l u s h i n g tubes were l e f t open, recovered i n the greenhouse,  although t h e i r growth was somewhat  slower than that of the p l a n t s that had no i c e - c o v e r ( F i g . 4 . 1 l ) . one of these p l a n t s a f t e r 10'days of growth i n the greenhouse, damage could be seen and v i t a l  On s e c t i o n i n g  no mechanical  s t a i n i n g revealed that a l a r g e p r o p o r t i o n o f  the c e l l s s t a i n e d red and were a l i v e ( F i g . 4.13). The p l a n t s , subjected to a 50-day ice-sheet cover, with the s o i l water-saturated, were k i l l e d .  Carbon d i o x i d e d i d not accumulate to l e v e l s  as high as i n the s o i l where the moisture was approximately at f i e l d probably due to the s o l u t i o n of the 00^  i n the water.  capacity,•  Since .the s o i l pore-  space was f i l l e d with water, only a s i n g l e gas sample could be withdrawn from the sampling tube, thus, the 0^ l e v e l s i n the pots could not be analyzed. A l l 4 p l a n t s when moved to the greenhouse f o r recovery, decayed  rapidly,  and could not be s e c t i o n e d . When s o i l , at approximately f i e l d capacity, was kept under a n - i c e sheet f o r - 5 0 days, CO^ accumulated to 5$ and 0^ was depleted to 10$ of the s o i l atmosphere.  Approximately h a l f of the CO,,, t h e r e f o r e , which  when forage p l a n t s are encased i n an i c e - s h e e t , may s o i l microorganism a c t i v i t y .  accumulated  probably be a t t r i b u t e d to  S o i l 0^ d e p l e t i o n on the other hand, i s u s u a l l y  a t t r i b u t e d to both chemical r e a c t i o n s and m i c r o b i a l r e s p i r a t i o n  (l09).  60  TABLE 4 . I I COMPOSITION OF THE SOIL ATMOSPHERE AFTER 50 DAYS AT -3.0°C. (i)  WITH ICE COVERED SOILS AT DIFFERENT MOISTURE LEVELS  ( i i ).  WITH ICE COVERED SOILS AERATED  (iii)  WITH ICE COVERED SOILS DEVOID OF PLANTS  Composition o f s o i l atm. (per cent) Dry wt. r o o t s f o l l o w i n g Treatment  Pot no.  Ice-sheet Soil F.C* Tubes c l o s e d  1 2 3 4  Ice-sheet Flooded Tubes c l o s e d  1 2 3 4  Ice-sheet S o i l F.C. Tubes open  1 2 3 4  No i c e - s h e e t Control  1 2  Ice-sheet No plant," S o i l F.C.  1 2  * **  F i e l d Capacity M i s s i n g data  0  CO2  2  4.2  8.2  3.7  10.0  -**  22.0 22.0 22.0 22>0  6.8  5080 4907 4853  9.0 3.3 3.3 2.4  2.00 1.85 1.95 1.70  4930 5361 5080 5334  0.28. 0.30 0.30 0.30  1.55 1.90 1.95 2.10  4994 5040 5384 4853  -—  5307 5361  —  5384 5307  Approx. atmospheric 7.2 12.8  s o i l (gm.)  1.95 2.10 1.80 1.70  9.8  , •  15 day recovery (gm.)-  - Dry wt.  6.0 4.0  61  Fig.  4.11  An ice-sheet l a s t i n g 7 weeks r e s u l t e d i n the death of the p l a n t s rooted i n both a saturated and i n a s o i l at approximately f i e l d c a p a c i t y . A e r a t i o n of the i c e covered s o i l by l e a v i n g the f l u s h i n g tubes open, prevented t o x i c accumulation of CC> .  Fig.  4.12  V i t a l s t a i n i n g of a s e c t i o n of a plant kept f o r 50 days at -3.0 C without ice-sheet cover. Sections were made f o l l o w i n g a 10-day recovery p e r i o d . s t a i n i n g c e l l s are i n d i c a t i v e of l i v e t i s s u e .  an Red  62  Fig.  4.15  V i t a l s t a i n i n g of a s e c t i o n of a plant covered by an ice-sheet f o r 50 days w i t h f l u s h i n g tubes l e f t open t o aerate the s o i l beneath the i c e . S e c t i o n s were made f o l l o w i n g a 19-day recovery p e r i o d . Many red s t a i n i n g c e l l s are visible. ( r ) red s t a i n i n g t i s s u e  Fig.  4.14  Vital soil kept Only  s t a i n i n g of a s e c t i o n of a p l a n t covered by an ice-sheet f o r 50 days. The moisture was a t approximately f i e l d c a p a c i t y and the f l u s h i n g tubes were c l o s e d . Carbon d i o x i d e accumulated t o over 10$ o f the s o i l atmosphere. a few red s t a i n i n g c e l l s could be d e t e c t e d , (y) y e l l o w s t a i n i n g t i s s u e  63 The manometers attached  to 2 pots ( F i g . 2.7) with s o i l approximately  at f i e l d capacity, and an ice-sheet on the surface, revealed an i n t e r e s t i n g pattern o f change i n pressure  with a change i n temperature.  "When the l i g h t  bulb i n the box with temperature c o n t r o l was switched o f f , a f r e e z i n g plane s t a r t e d progressing downward, with a subsequent build-up i n pressure the frozen s o i l .  beneath  When the l i g h t bulb was- switched on again, the lower end o f  the pots i n s i d e the box were warmed, and the pressure a r e t r e a t of.the- f r e e z i n g plane.  dropped as a r e s u l t o f  This pattern proved t o be very u s e f u l i n  c o n t r o l l i n g the temperature i n the i n s u l a t e d boxes, by merely observing the pressure.  4.4..  4.4.1.  The extent  -EXPERIMENT IY  to which a freeze-thaw-freeze c y c l e , a s s o c i a t e d with an  i c e - s h e e t , i s damaging t o a l f a l f a . 4.4.2.  Does stubble, protruding through an ice-sheet, serve as a passage f o r gas d i f f u s i o n t o the root system, and i n t h i s way be o f some p r o t e c t i o n against ice-sheet damage?  A l t e r n a t e f r e e z i n g and thawing, a s s o c i a t e d with-an ice-sheet was not. damaging t o the plants..  The 4 p l a n t s that were subjected  to a 20-day  ice-sheet cover, followed by'-a 10-day thaw p e r i o d , and then another 10-day ice-cover, recovered  almost as s t r o n g l y as the p l a n t s exposed t o the same  temperatures, but which were not covered by an. ice-sheet ( F i g . 4.16). 8 p l a n t s subjected the treatment.  to a continuous, ice-sheet f o r 40 days, b a r e l y  The  survived  During the 10-day thaw p e r i o d , temperatures under the  TABLE 4. I I I . COMPOSITION OF THE SOIL ATMOSPHERE AFTER 40 DATS AT - % 0 ° C . (i)  CONTINUOUS ICE-COVER WITH PLANTS ENTIRELY COVERED AND WITH STUBBLE PROTRUDING THROUGH THE ICE  (ii)  ICE-COVER INTERRUPTED BY A 10 DAY THAW PERIOD  Composition o f s o i l atm. (per cent)  co 2  °2 Treatment  Pot no.  20 10 days days  Fr-Thaw-Fr** 20-10-10*** Ice-sheet  1 2 3 4  Fr-Thaw-Fr 20-10-10 No ice-sheet Control  1 2 3 4  Continuous i c e Entirely covered  1 2 3 4  7.0 1.8  1 2 3 4  ___  Continuous i c e Stubble i  *  M i s s i n g data Freeze-Thaw-Freeze Days  4.0 1.9 • -* -  4.0 2.0 ' 5.0 5.2  20 10" days days 5.4 4.6 2.8 3.4  3.0 2.8 3.2 3.8  D  5?Iay Roots 2.15 2.85 1.85 2.00  Approx. atmospheric  —1.8  1.8 4.8 4.6  plant f o l l o w i n g recovery (gm.;. Tops  Tot. 6.30  Tot. 7.85  5o8 5.8 8.8 7o6  3.00 1,85 2.80 lo80  7.2 9.2 5.6 5.6  3.50 2.15 1.90 1.55  Tot. 2.15  Tot. 2.35  " Dry wt. • s o i l (gm.) 5448 5534 5221 5448 5675 5280 5675 5307 5448 5248 5734 5507 5448 5534 5361 5193  65 thawing cover ranged from 5° to 15°C. The i c e melted r a p i d l y , i n c r e a s i n g the  s o i l moisture considerably, and the winter dormancy o f the a l f a l f a  broken.  Fig.  was  New shoots emerged, and a f t e r 10 days were 15 to 20 cm. high ( F i g . 4.15).  4.15  Growth of new shoots a t the end of a 10-day period under the thawing-cover. P r i o r to the thawing period, the plants on the l e f t were covered by an i c e sheet f o r 20 days, while the ones on the r i g h t were exposed t o the same f r e e z i n g temperature, but had no i c e - c o v e r .  Stubble protruding through the i c e , seemingly, d i d not help the plants to withstand long periods o f ice-sheet cover.  With the techniques  employed, passage of gases through the stubble t o and from the root system could not be detected. ice,  In the pots, where stubble was protruding through the  CC^ accumulated to almost the same l e v e l as i n the pots where the p l a n t s  were e n t i r e l y encased. error.  The d i f f e r e n c e was w e l l w i t h i n the range o f experimental  66  Fig.  4.16  Due t o an i n t e r r u p t i o n i n the i c e - c o v e r hy a thaw p e r i o d , CO,, d i d not accumulate t o the same extent as i n the pots which were c o n t i n u o u s l y covered by an i c e - s h e e t , and as a r e s u l t , the thaw appears t o have been b e n e f i c i a l r a t h e r than more damaging t o the p l a n t s . Stubble p r o t r u d i n g through the i c e , apparently d i d not help the p l a n t s t o withstand l o n g periods o f i c e - c o v e r .  Fig.  4.17  V i t a l s t a i n i n g of a s e c t i o n taken just below the crown of a plant exposed to a 20-day f r e e z e , 10-day thaw and a 10-day freeze period, without any i c e - c o v e r . Sections were made a f t e r 8 days recovery i n the greenhouse.  67  F i g . 4.18 V i t a l s t a i n i n g o f a s e c t i o n of a plant exposed to a 20-day f r e e z e , 10-day thaw and a 10-day freeze period, with an ice-sheet cover during the two freeze periods. Sections were made a f t e r 8 days recovery i n the greenhouse.  F i g . 4.19 V i t a l s t a i n i n g of a s e c t i o n taken j u s t below the crown of a plant encased f o r 40 days i n an ice-sheet, with the stubble protruding through the i c e . Sections were made a f t e r an 8-day recovery p e r i o d .  68  Fig.  4.20  Y i t a l s t a i n i n g o f a s e c t i o n taken j u s t below the crown o f a p l a n t e n t i r e l y encased i n an i c e - s h e e t w i t h no p a r t s p r o t r u d i n g through the i c e .  4.5.  EXFERDffiMT Y  Does a h i g h s o i l moisture content i n the w i n t e r r e s u l t i n an i n c r e a s e d h y d r a t i o n l e v e l i n the t i s s u e which i n t u r n i n c r e a s e s the p l a n t s ' s u s c e p t i b i l i t y to freezing? No i n c r e a s e could be detected i n the moisture l e v e l i n the t i s s u e of the p l a n t s kept f o r 12 days i n a near f r e e z i n g , water s a t u r a t e d s o i l ( F i g . 4 . 2 l ) . Furthermore, when these p l a n t s were exposed t o a -10.0°C temperature, the i n j u r i e s s u f f e r e d by the p l a n t s kept i n a s a t u r a t e d s o i l were no g r e a t e r than the i n j u r i e s s u f f e r e d by those kept i n a d r y s o i l , and those kept i n a s o i l near f i e l d c a p a c i t y ( F i g . 4.22).  The d i f f e r e n c e i n the q u a n t i t y o f amino a c i d leached  from each o f the three groups o f p l a n t s , t h a t had been kept i n s o i l s a t the  69 E F F E C T  OF  ON  M O H S T U R E  -CONSE  ON  T B S S U E  E X T E N T  THE  MOISTURE C O L D  OF  91-2  90  &  9 0 82-2 81-4  807522  73-96  74.87  V,  70-  >  3  60-  z  50-  > n  o  I-  45-68  (/) O  2  o  s: •  40-  3214  %  30-  1  3,...  I* .A  2015-80  is ' <  10 -  t -i J X x  •"<fl  :  i  I0RY  ~ FIELD CAP.  SOIL  ROOT  FIG  4.21  SATURATED  AMINO ACID  /« m o l s s / g m .  70  three d i f f e r e n t moisture l e v e l s , i s s t a t i s t i c a l l y not s i g n i f i c a n t .  (c.) Fig.  4.22  ( a . ) , (t>.), and (c.) — response of sections of plants stored f o r 48 hours at  71 -10.0 C to v i t a l s t a i n i n g . P r i o r to the f r e e z i n g treatment the plants were kept f o r 12 days a t near f r e e z i n g temperature i n a saturated s o i l ( a . ) , a s o i l a t approximately f i e l d c a p a c i t y ( b . ) , and a dry s o i l ( c ) . Sections of the plants exposed to s o i l s at d i f f e r e n t moisture l e v e l s revealed some l i v e t i s s u e , mostly dead t i s s u e and some mechanical d i s r u p t i o n . There appears to he no c l e a r c o r r e l a t i o n between the amount of damage to the plants and the moisture l e v e l of the s o i l .  4.6.  EXPERIMENT VI  Is CC^ accumulation o r 0^ d e p l e t i o n or both, the causal f a c t o r i n ice-sheet i n j u r y ? The p l a n t s rooted i n a s o i l which was flushed with e i t h e r  °  T  CO^-free a i r , while covered by an ice-sheet f o r 21 days, recovered v i g o r o u s l y i n the greenhouse.  On the other hand, the 2 p l a n t s that had CO^ flushed  through the s o i l while covered by i c e , d i d not survive ( F i g . 4.23).  Carbon  d i o x i d e accumulation, r a t h e r than 0^ d e p l e t i o n was, therefore, the causal f a c t o r i n ice-sheet i n j u r y .  Fig.  4.25  Carbon dioxide accumulation r a t h e r than 0^ d e p l e t i o n was responsible f o r the damage to the i c e covered a l f a l f a . P l a n t s rooted i n a s o i l through which N ^ was passed almost continuously, s u f f e r e d no i n j u r y .  72 The  COg l i b e r a t e d by s o i l microorganisms and plant r o o t s , when Og,  Ng and COg - f r e e a i r was passed through the i c e covered s o i l , was c o l l e c t e d on " A s c a r i t e " which was weighed p e r i o d i c a l l y . The weights o f COg l i b e r a t e d are presented i n TABLE  6.IV.  TABLE 6.17. THE WEIGHTS OF C 0 LIBERATED WHEN ICE COVERED SOILS WERE FLUSHED WITH N , Og, 2  AND  C0 -FREE AIR. 2  F l u s h i n g gas Time f l u s h e d Weight s o i l (gm.) N  2  °2 Air-COg  Weight roots (gm.)  COo l i b e r a t e d (gm.)  96 h r s . •  12031  5.85  0.2827  96  hrs.  11804  8.15  0.1857  96 h r s .  11690  7.60  0.7768  High concentrations  o f both Og and Ng appeared t o have had an  i n h i b i t o r y e f f e c t on root and s o i l organism a c t i v i t y since f a r l e s s COg was l i b e r a t e d when the s o i l was f l u s h e d with e i t h e r o f the two gases, than with COg  - free a i r .  S l i g h t l y more COg was l i b e r a t e d when Ng , than when Og was  passed through the s o i l .  The d i f f e r e n c e i s s t a t i s t i c a l l y  4.7.  The  not  significant.  EXPERIMENT VII  r e a c t i o n o f s e v e r a l species and v a r i e t i e s o f forage p l a n t s t o  withstand a prolonged period o f i c e encasement.  A l l v a r i e t i e s and species t e s t e d were a f f e c t e d t o a greater o r l e s s e r extent, as a r e s u l t o f being e n t i r e l y encased i n i c e f o r a 2-month p e r i o d . The  performance o f the d i f f e r e n t v a r i e t i e s and species i s summarized i n TABLE 7.V.  7 3  TABLE 7.V. SURVIVAL. OF DIFFERENT SPECIES AND VARIETIES FOLLOWING ICE ENCASEMENT FOR 6 0 DAYS  P l a n t species o r v a r i e t y  Winter* No. o f plants hardiness growing Ice No i c e rating'  Orchardgrass ( D a c t y l i s glomerata) V a r i e t y Hercules " Danish " Chinook " Latar' " Rideau  W.H. W.H. W.H. N.W.H. W.H.  A l f a l f a (Medicago s a t i v a ) V a r i e t y Lahontan " Ladak " Rambler " Beaver " C a l i f o r n i a Common " Rhizoma  N.W.H. W.H. W.H. W.H." N.W.H. F.W.H.  Dry weight following 2 weeks recovery (gm.) No i c e Ice  0  4  0  0 . 7 6 2  0  3  0  0 . 6 2 9  0  4  0  0 . 7 3 5  0  4  0  0 o 4 5 3  3  4  0 . 1 8 1  0 . 7 1 0  1  4  0 . 0 0 6  0 . 4 6 2  1  4  0 . 0 0 2  0 . 2 6 8  2  3  0 . 0 6 1  0 . 3 4 9  2  4  0 . 2 5 4  0 . 4 7 3  0  4  0  0 „ 4 1 3  0  3  0  0.230  0  0  1  1  0.00.4  0 o 0 1 2  -  Sweet Clover ( M e l i l o t u s alba) V a r i e t y Denta " Common White Blossom  W.H. W.H.  Red Fescue Penlawn (Festuca rubra)  W.H.  4  4  0 . 8 5 0  1 . 1 0 4  F.W.H.  1  4  0 . 0 0 5  0 . 5 9 5  W.H.  4  4  0 . 9 7 8  P e r e n n i a l Rye Grass (Lolium perenne)  N.W.H.  0  4  0  0 o 4 6 8  New Zealand Wild White Clover ( T r i f o l i u m repens)  F.W.H.  0  3  0  0 . 2 4 4  A l s i k e Clover Aurora' ( T r i f o l i u m hybridum)  W.H.  0  1  0  0 „ 1 1 0  •Winter hardiness r a t i n g  W.H.  Highland'Bent l A g r o s t i s sp.) Reed Canary Grass ( P h a l a r i s arundinacea)  = Winter hardy  F.W.H. = F a i r l y winter hardy N.W.H. = Non winter hardy  0  0  '  1 . 3 4 6  74  5.  DISCUSSIONS  In c o n s i d e r i n g the r e s u l t s from the experiments * i t i s necessary to  emphasize the o b j e c t i v e o f the study.  The aim was to provide information  on t h e e f f e c t of an i c e - s h e e t , -at non-injurious temperatures, .on the compo-  s i t i o n o f t h e . s o i l atmosphere, and t o determine the nature o f the damage s u f f e r e d by overwintering p l a n t s rooted i n the i c e covered s o i l . The study was conducted under c o n t r o l l e d environmental c o n d i t i o n s , due to the sporadic occurrence o f i c e - s h e e t s i n nature.  To simulate n a t u r a l  i c e - s h e e t s , which u s u a l l y p e r s i s t f o r many weeks or even months, .each experiment r e q u i r e d a r a t h e r long time to complete.  Furthermore, the study  was somewhat complicated- by the s i z e and complexity of the apparatus as w e l l as t h e . l i m i t e d space i n the f r e e z i n g room. Under a r t i f i c i a l Ice-sheets as used i n t h i s study, COg accumulated i n some instances t o as h i g h as 1 0 $ , while 0  o  was-depleted•to 3 . 7 $ of the  75 s o i l atmosphere.  Such conditions developed under an i c e - c o v e r l a s t i n g 50  days, and r e s u l t e d i n the death of the overwintering p l a n t s . s h o r t e r durations, r e s u l t i n g i n a lower accumulation of CO^ caused i n j u r y to the p l a n t s .  Ice-sheets  of  and d e p l e t i o n of  Both death and i n j u r y were p h y s i o l o g i c a l  r a t h e r than mechanical. Under n a t u r a l f i e l d c o n d i t i o n s , a s i m i l a r , f a i r l y r a p i d accumulation of COg under an ice-sheet, could he expected. increases with depth (20, 145)  and  Since the s o i l CO^  since the formation  concentration  of ice-sheets i s  u s u a l l y accompanied hy high s o i l moisture, a c e r t a i n .amount of the CO^ at g r e a t e r depths could be d i s p l a c e d upward by water p e r c o l a t i n g downward. CO^ would be trapped by the ice-sheet on the surface of the s o i l , and way  could contribute to i t s accumulation i n the r o o t i n g zone of the The methods a v a i l a b l e f o r determining  CO^  The  this  soil.  i n the s o i l do not  allow  measurement of concentrations at the immediate root surface, but mainly represent CO^  concentrations  of the l a r g e r s o i l pores.  pointed out by e a r l i e r workers ( l 9 , 55). f i l m surrounding  The CO^  T h i s f a c t has been  concentration i n the water  the root can be expected to be higher, and 0^  concentration  lower, than i n the s o i l atmosphere due to the much higher, s o l u b i l i t y d i f f u s i o n r a t e of  and  CCy  B r i e r l e y et a l . (22) i n an e f f o r t to determine the composition the atmosphere immediately surrounding  an i c e encased p l a n t , t i e d  of  strawberry  p l a n t s to the e x t e r i o r of screen -wire c y l i n d e r s , and then coated the p l a n t s and the c y l i n d e r s with i c e .  Following 3 weeks under such c o n d i t i o n s , which  are q u i t e removed from the n a t u r a l s t a t e , they analyzed  the gases w i t h i n the  c y l i n d e r s and found that CO,, accumulated to concentrations as high as while  d e c l i n e d to about 4$, and that the p l a n t s were k i l l e d by  treatment.  The highest CO  concentration of 10$ recorded  24$,  this  i n the present  study  76 i s considerably lower than the concentration recorded by B r i e r l e y et a l . (22). The Og on the other hand was s o i l atmosphere. disappearance  depleted to as low or even lower than 4$ of the  The f i n d i n g s of Scott and Evans ( l l 5 ) concerning the r a p i d  of d i s s o l v e d Og i n saturated s o i l s may be a p p l i c a b l e , to a  c e r t a i n extent, to moist s o i l s such as were used i n i c e - s h e e t experiments reported here.  The gas analyses made by B r i e r l e y et a l . (22) u s i n g screen  c y l i n d e r s as w e l l as the analyses of Sprague and Graber (125, 126), and Dale Smith ( l 2 0 , 12l) u s i n g sealed t e s t tubes, c o n s t i t u t e a l l attempts made to date to assess the e f f e c t of an ice-sheet on the composition of the atmosphere surrounding the p l a n t . The gas-chromatogram used i n t h i s study was and CH^,  capable of s e p a r a t i n g CO  however, n e i t h e r of the two gases was found to accumulate under an  ice-cover.  Cyanide  (HCN), on the other hand was not measured and i n some  cases a b s o r p t i o n of the compound by the crown t i s s u e may a s s o c i a t e d with i c e - s h e e t s .  Cyanide i s produced  result i n injury  during the m y c e l i a l growth  of an u n i d e n t i f i e d basidiomycete r e s p o n s i b l e f o r winter crown r o t .  This  disease i s frequently confused with other forms of winter k i l l i n g and i s p a r t i c u l a r l y v i r u l e n t and i n j u r i o u s to a l f a l f a and c l o v e r s .  Infection i s  most severe under c o n d i t i o n s of a slowly m e l t i n g snow cover and i s a l s o i n fluenced by s o i l moisture,(33).  A period of 45 to £0 days of a s s o c i a t i o n  between the host and pathogen i s r e q u i r e d f o r the absorption.of l e t h a l concentrations of HCN by the host, and mass i n v a s i o n does not occur u n t i l the t i s s u e s had absorbed  l e t h a l q u a n t i t i e s of HCN  (70, 7 l ) . No s t u d i e s have yet  been made on the e f f e c t of s o i l a e r a t i o n on- disease development.  I f the  winter crown r o t basidiomycete, d i d p l a y a r o l e i n the i n j u r y of the p l a n t s used i n the simulated i c e - s h e e t s t u d i e s , then the p a t h o g e n i c i t y of the fungus must be s t r o n g l y dependent on the composition of the s o i l atmosphere, since  77 the r e s u l t s of the experiments  show that damage was always a s s o c i a t e d  with high COg l e v e l s . When the r a t e and the l e v e l t o which COg accumulates under an i c e sheet was accentuated by f l u s h i n g the i c e covered s o i l with the gas, overw i n t e r i n g p l a n t s were k i l l e d l e v e l s the a l f a l f a  was k i l l e d  by t h i s treatment.  With such induced h i g h COg  w i t h i n 21 days, compared to approximately  50 days o f i c e - c o v e r necessary to k i l l p l a n t s without an a r t i f i c i a l l y modified s o i l atmosphere.  When the s o i l was aerated o r f l u s h e d with Og, the p l a n t s  s u f f e r e d no apparent damage, even a f t e r being covered by i c e f o r 62 days. The i n j u r i o u s e f f e c t o f COg as an e x t e r n a l medium i s not a recent o b s e r v a t i o n .  As e a r l y as 1804, de Saussure, c i t e d by Clements (32)  observed that growing chestnut p l a n t s , whose r o o t s were stored i n COg, d i e d i n 7 to 8 days, while those stored i n a i r were s t i l l vigorous at the end o f 3 weeks.  Cornwinder,  a l s o c i t e d by Clements (32), found that marsh p l a n t s  died q u i c k l y when t h e i r r o o t s were submerged i n water charged with COg. Free (46) observed the same r e l a t i o n i n buckwheat. stored a l f a l f a  Sprague and Graber (l26)  r o o t s and crowns a t -4.0°C i n a t e s t tube through which COg was  passed, and found that p l a n t s surrounded by 25$ and 50$ COg were s e r i o u s l y weakened a f t e r 21 days and were dead a f t e r 54 days.  .Although the experimental  techniques used by Sprague and Graber ( l 2 5 , 126) subjected the p l a n t s t o c o n d i t i o n s that a r e somewhat removed from those found under n a t u r a l i c e - s h e e t s , t h e i r r e s u l t s c l e a r l y i n d i c a t e that high COg l e v e l s are i n j u r i o u s t o dormant alfalfa  a t a s u b - f r e e z i n g temperature. The p h y s i o l o g i c a l damage to the t i s s u e of the p l a n t s covered by  ice,  with an a r t i f i c i a l l y high COg l e v e l surrounding the underground p a r t s ,  could not be a t t r i b u t e d to a change i n pH o f the s o i l .  The s o i l used  appears  to have been s u f f i c i e n t l y b u f f e r e d to prevent a lowering of i t s pH by s o l u t i o n  78 of COg i n the s o i l water» 1.0°C  A f t e r i n t e r m i t t e n t l y f l u s h i n g a wet s o i l kept a t  with COg f o r 2 weeks, no change i n the s o i l pH could he detected.  The  damage to the t i s s u e , t h e r e f o r e , appears t o be due to t o x i c l e v e l s of COg, o r a l a c k of Og, or both. When an i c e covered s o i l was f l u s h e d with e i t h e r Og, Ng, o r COg-free a i r f o r 21 days, the p l a n t s s u f f e r e d no damage and grew w e l l i n the greenhouse. COg  On the other hand when an i c e covered s o i l was f l u s h e d with  f o r the same period o f time, the p l a n t s were k i l l e d .  T h i s r e s u l t shows  that COg accumulation r a t h e r than Og d e p l e t i o n i s the causal f a c t o r i n i c e sheet i n j u r y . Sprague and Graber (l26) u s i n g a s l i g h t l y d i f f e r e n t experimental approach, a l s o found that only COg, as an e x t e r n a l medium, was d i r e c t l y harmful to a l f a l f a  plants.  They found that dormant a l f a l f a  p l a n t s stored  at noninjurious temperatures o f -3.5°C' and -4.0°C f o r v a r y i n g periods i n t e s t tubes of e x t e r n a l media which removed COg such as flowing a i r and Ng sustained much l e s s i n j u r y then those confined f o r s i m i l a r periods i n t e s t tubes through which COg was passed. It i s d i f f i c u l t  t o conclude from a l l these r e s u l t s that s u f f o c a t i o n  i s the major f a c t o r responsible f o r ice-sheet damage i n the f i e l d .  Under  f i e l d c o n d i t i o n s , where numerous f a c t o r s are u s u a l l y a s s o c i a t e d with winter i n j u r y , i t i s p o s s i b l e that i c e - s h e e t s may cause death d i r e c t l y from c o l d , e s p e c i a l l y since i c e has a thermal c o n d u c t i v i t y f o u r times as great as water and about a 100 times that o f a i r . i n d i c a t e that a t noninjurious  However, the r e s u l t s of t h i s study c l e a r l y  low temperatures t o x i c l e v e l s o f COg d i d  accumulate under p e r s i s t i n g i c e - s h e e t s and could cause extensive overwintering  forage  damage to  crops.  Many workers have found that Og at a pressure  o f 1 atmosphere o r  79 lower has i n j u r i o u s e f f e c t s on p l a n t s (29, 104, 137).  E l i a s s o n (44) has  found t h a t Og a t a pressure s l i g h t l y lower than 1 atmosphere completely i n h i b i t e d the growth o f wheat r o o t s w i t h i n 3 days, and t h a t recovery was slow and incomplete.  High l e v e l s o f Og i n the i c e covered s o i l had an  i n h i b i t o r y e f f e c t on root and s o i l - o r g a n i s m r e s p i r a t i o n , s i n c e f a r l e s s COg was l i b e r a t e d when the s o i l was f l u s h e d w i t h Og than w i t h COg-free a i r . The p l a n t s , however, d i d not s u f f e r any "oxygen-poisoning" and when moved t o the greenhouse they grew as v i g o r o u s l y as those t h a t had a i r f l u s h e d through the s o i l .  Thus,  h i g h - l e v e l s o f .Og appear t o be t o x i c t o a c t i v e l y m e t a b o l i z i n g  p l a n t s , r a t h e r than t o dormant p l a n t s e s p e c i a l l y a t s u b - f r e e z i n g temperatures. The 4 p l a n t s rooted i n a water s a t u r a t e d s o i l and covered by an i c e sheet f o r 50 days were k i l l e d , and decayed r a p i d l y when subsequently moved t o the greenhouse. Sprague and Graber ( l 2 6 ) s t o r e d 'Turkestan' a l f a l f a r o o t s i n a stoppered t e s t tube f i l l e d w i t h water a t 1.0°C, and found t h a t unfrozen water produced the same d e t r i m e n t a l e f f e c t as i c e , and the recovery o f the p l a n t s s t o r e d i n water c l o s e l y resembled those o f p l a n t s stored i n i c e , but water s a t u r a t e d w i t h COg caused the g r e a t e s t harm and a l l o f the p l a n t s were s e v e r e l y i n j u r e d i n 23 days.  Beard (6) submerged whole t u r f plugs o f 'Toronto'  bentgrass ( A g r o s t i s p a l u s t r i s ) , annual b l u e g r a s s (Poa annua), and Kentucky b l u e g r a s s (Poa p r a t e n s i s ) i n water, a l s o h e l d a t 1.0°C and found t h a t a l l 3 species s u r v i v e d w e l l even a f t e r 90-day p e r i o d s i n the water.  He suggested  t h a t Og may have been present i n the water f o r the r e s p i r a t i o n requirements of the p l a n t s .  The f i n d i n g s o f  Scott  and Evans ( l l 5 ) , - a l m o s t 10 years e a r l i e r ,  c o n t r a d i c t the suggestions put forward by Beard (-6).  The former two workers  found t h a t d i s s o l v e d Og s t a r t e d t o decrease immediately a f t e r the s o i l was f l o o d e d , and w i t h i n 10 hours i t had disappeared e n t i r e l y .  A f t e r the 0  o  was  80 depleted,-more 0,, was i n f r e s h l y flooded The  added, which disappeared even more r a p i d l y than i t d i d  soils.  d i s s o l v e d C ^ - i n - both Beard's (6)  and Sprague and  experiments, as w e l l as i n the ice-sheet covered saturated i n t h i s l a b o r a t o r y , must have been depleted  rapidly (l09).  s o i l , accumulation of the gas was  f a c t o r i n the i n j u r y to the p l a n t s . t u r f plugs submerged i n water (6), accumulate to t o x i c l e v e l s .  Since r e s p i r a t o r y  to 5$ and  0^ was  of the CO,,,  probably the  could escape and most l i k e l y  Such f i n d i n g s suggest that 00^  causal the  d i d not  accumulation,  plants.  When s o i l , at approximately f i e l d c a p a c i t y , and p l a n t s were rooted, was  ice-sheet  On the other hand CO,, l i b e r a t e d by  r a t h e r than 0^ d e p l e t i o n i s i n j u r i o u s to overwintering  (126)  s o i l experiment  CO,, could not escape from the stoppered t e s t tubes nor from the covered water saturated  Graber's  in  which no  kept under an ice-sheet f o r 50 days, CO,, accumulated  depleted  to 10$ of the s o i l atmosphere.  Approximately h a l f  t h e r e f o r e , which accumulates when forage p l a n t s are encased i n  an i c e - s h e e t , may  probably be a t t r i b u t e d to s o i l microorganism a c t i v i t y .  S o i l 0^ d e p l e t i o n on the other hand, i s a t t r i b u t e d to both chemical r e a c t i o n s and  to m i c r o b i a l r e s p i r a t i o n (l>15)'.  No  c l e a r separation of the m i c r o b i a l  and  chemical demands f o r 0,, has yet been made.  Scott and Evans ( l l 5 )  observed, however, that the r a t e of 0^ consumption i s g r e a t l y reduced i f the s o i l ' i s  sterilized.  Beard (8) a f t e r a s e r i e s of experiments, suggested that combinations of f r e e z i n g and l e v e l s may  thawing p o s s i b l y i n a s s o c i a t i o n with high plant t i s s u e moisture  be of g r e a t e r • s i g n i f i c a n c e i n w i n t e r i n j u r y a s s o c i a t e d with i c e -  sheets than d i r e c t 0,, s u f f o c a t i o n or t o x i c accumulations.  The  r e s u l t s of the  freeze-thaw-freeze experiment i n t h i s l a b o r a t o r y c o n t r a d i c t Beard's I t was  found that due  to the thaw p e r i o d , 0  was  not depleted  and  C0  suggestions. o  d i d not  81 accumulate to the same extent as i n the pots which were continuously  covered  by an i c e - s h e e t , and as a r e s u l t , t h e r e f o r e , the thaw appears to have been b e n e f i c i a l r a t h e r than more damaging to the plants.(Table 4 . I I I ) . shoots were k i l l e d during the second freeze p e r i o d . not s u f f e r any  The  The  new  crowns, however, d i d  extensive damage, in- s p i t e of the f a c t that t h e i r dormancy had  been broken which would have made tham more s u s c e p t i b l e to c o l d i n j u r y ('Fig.--4.18). Beard ( 7 ) compaction and He  reported that the- formation  of a s l u s h followed  f r e e z i n g i s - extremely damaging to some p e r e n n i a l  by  grasses.  suggested that the compacting a c t i o n reduced a i r pockets i n the sod, which  i n t u r n could r e s u l t i n an increased h y d r a t i o n l e v e l i n the t i s s u e due increased' contact between the p l a n t tissue- and  the'slush.  to  Increased t i s s u e  moisture content r a i s e s the k i l l i n g temperature, with mechanical d e s t r u c t i o n ' r e s u l t i n g from the formation  of l a r g e i c e masses.  A number of: workers have pointed out that the r a t e . o f water by the plant i s dependent on the s o i l temperature ( 6 6 , summarized .in F i g . 4 . 2 1 , 149),  and  149).  The r e s u l t s  agree with the f i n d i n g s reported e a r l i e r (66.,  103,  indicate^ that at- low temperatures the plants d i d not absorb water  r e a d i l y , no matter how  moist was'the s o i l .  were.frozen, those that were kept i n a wet those that were-kept i n a dry s o i l . by others  103,  absorption  (66,  a s l u s h may  103,  149)  Furthermore, when the same p l a n t s s o i l "suffered no more damage than  This' f i n d i n g , i n :'addition to those  reported  made by Beard ( 7 ) ,  i s contrary to the suggestion  that  be r e s p o n s i b l e f o r an increased h y d r a t i o n l e v e l i n the plant t i s s u e .  The  a b i l i t y of s e v e r a l v a r i e t i e s and  stand a 2-month period of i c e encasement was  species of forage  tested.  p l a n t s to  A l l v a r i e t i e s and  t e s t e d were a f f e c t e d to a g r e a t e r - o r l e s s e r extent, and  on the whole, no  d e f i n i t e c o r r e l a t i o n was- found between winter hardiness  and  the d i f f e r e n t p l a n t s to withstand ice. encasement.  with-  species  the a b i l i t y of ;  82 Sjoseth ( l l 9 ) i n h i s s t u d i e s on i c e encasement of s t r a i n s of red c l o v e r ( T r i f o l i u m pratense) and timothy (Phleum pratense) used a very s i m i l a r technique employed i n the t e s t i n g of the v a r i e t i e s and species l i s t e d i n Table 7.V.  In red c l o v e r , Sjoseth ( l l 9 ) found that d i f f e r e n t i a l s u r v i v a l  f o l l o w i n g i c e encasement occurred, but that there was no c l e a r - c u t between r e s i s t a n c e to i c e encasement and f r o s t hardiness.  correlation  With timothy, however,  he found that d i f f e r e n t i a l ' s u r v i v a l corresponded w e l l with the g e n e r a l l y known winter hardiness of the s t r a i n s .  Beard  (8) employing  a s i m i l a r technique, found  that while Kentucky bluegrass and annual bluegrass were k i l l e d at the end of 45 days of i c e encasement, 'Toronto' bentgrass e x h i b i t e d 100$ t h i s adverse treatment  through the f i r s t 60 days.  s u r v i v a l under  In a l a t e r study Beard  (6)  t e s t e d d i f f e r e n t bentgrass v a r i e t i e s , and found that as a group, the bentgrasses were quite t o l e r a n t to extended  i c e coverage.  As i n d i c a t e d e a r l i e r , the  technique of encasing e n t i r e ^ p l a n t s used by Beard (6,8)  and Sjoseth ( l l 9 )  r e s u l t s i n the build-up of a h y d r o s t a t i c pressure toward the center of the potted s o i l as the f r e e z i n g plane progresses from a l l d i r e c t i o n s .  Such an unnatural  c o n d i t i o n has been avoided i n the present study by embedding i n sand the potted p l a n t s to be f r o z e n .  The sand, i n a w e l l i n s u l a t e d box, provided enough  i n s u l a t i o n , so that, the f r e e z i n g plane progressed only v e r t i c a l l y downward, without a b u i l d - u p i n pressure. B r i e r l e y et a l . (22) and L a r i n (69) f r o z e n , waterlogged  studied the p e r m e a b i l i t y of  s o i l s and found that very l i t t l e gaseous d i f f u s i o n takes  place through them, while o r d i n a r y f i e l d s o i l s with a normal water content, when frozen, were found to.be q u i t e permeable. The strong recovery of the potted p l a n t s - t h a t were rooted i n a s o i l without excessive moisture, and that were not surrounded by i c e , suggests that gases r e a d i l y permeated to the root system of these p l a n t s .  On the other hand  83 the poor recovery o f the p l a n t s that were completely surrounded by i c e , and t h a t were rooted i n a s o i l a t the same moisture l e v e l and kept a t the same temperature f o r the same p e r i o d o f time as those p l a n t s t h a t were not encased i n i c e , suggests t h a t the i c e l a y e r acted as a b a r r i e r t o the normal d i f f u s i o n of gases. Of a l l the v a r i e t i e s and species t e s t e d , r e d fescue (Festuca r u b r a ) , which i s g e n e r a l l y considered t o be a hardy g r a s s , and reed  canarygrass  ( P h a l a r i s arundinacea) were best able t o withstand the 2-month i c e encasement. Reed canarygrass, i n most cases, grows on p o o r l y drained s o i l s , f l o o d i n g and s i l t i n g .  subjected t o  B o l t o n ( l 7 ) found t h a t 49 days o f f l o o d i n g d i d not  .cause excessive permanent-injury  to t h i s grass.  Under such moist c o n d i t i o n s ,  the stagnant water o f t e n f r e e z e s i n the w i n t e r forming an i c e - s h e e t . hydrophytes a r e equipped w i t h organs such as aerenchymatous t i s s u e , which Og d i f f u s e s from the shoots t o the root system.  Normally through  Many p l a n t s , however,  such as the monocotyledons, do not possess aerenchymatous t i s s u e and the mechanism o f gas transport- i s not c l e a r l y understood.  Yet s u f f i c i e n t data  have been c o l l e c t e d t o demonstrate t h a t there i s ample f a c i l i t y f o r Og t o d i f f u s e down p l a n t s when only the normal i n t e r c e l l u l a r spaces a r e present  (l46).  Since reed canarygrass s u r v i v e d a 2-month p e r i o d e n t i r e l y encased i n i c e w i t h no shoots p r o t r u d i n g , i t would appear t h a t i t most probably i s able not o n l y t o aerate i t s r o o t s v i a the shoots, but a l s o t o withstand f a i r l y h i g h COg l e v e l s that.may have developed w i t h i n the f r o z e n p o t . No c l e a r - c u t c o r r e l a t i o n between r e s i s t a n c e t o i c e encasement and f r o s t h a r d i n e s s could be found i n the orchardgrass ( P a c t y l i s glomerata) v a r i e t i e s tested.  Of the 4 v a r i e t i e s t h a t d i d not withstand i c e encasement, o n l y 'Latar'  i s non-hardy, w h i l e 'Hercules','Danish', bred f o r w i n t e r h a r d i n e s s .  and p a r t i c u l a r l y 'Chinook' have been  The only v a r i e t y t o s u r v i v e the-treatment was  'Rideau',. which i s considered t o be very w i n t e r hardy.  84 The d i f f e r e n t i a l recovery of a l f a l f a (Medicago s a t i v a )  following i c e  encasement, corresponds more c l o s e l y to the g e n e r a l l y known winter hardiness of the same s t r a i n s , than does the recovery of orchardgrass. and p a r t i c u l a r l y the treatment.  1  Rhizoma', 'Lahontan',  ' C a l i f o r n i a common' are not winter hardy and d i d not survive 'Ladak', :'Rambler', and e s p e c i a l l y 'Beaver' are a l l known f o r  t h e i r winter hardiness and survived the i c e encasement f a i r l y w e l l , with the exception o f 'Ladak', which was badly damaged by the treatment. .Sweet c l o v e r ( M e l i l o t u s alba) i s w e l l known f o r i t s a b i l i t y to grow and g e n e r a l l y survive a v a r i e t y o f adverse c o n d i t i o n s . Yet the coumarin-free v a r i e t y 'Denta' d i d not t o l e r a t e even a 2-month period at -3.0°C, without being encased  i n i c e . -The common white blossom sweet c l o v e r d i d not f a r e much b e t t e r .  T h i s observation i s r a t h e r s u r p r i s i n g e s p e c i a l l y since, of a l l the v a r i e t i e s and species t e s t e d , the sweet c l o v e r s together with a l s i k e c l o v e r ( T r i f o l i u m hybridum) were damaged only as a r e s u l t of storage at -3.0°C. Highland Bent ( A g r o s t i s spp.) and e s p e c i a l l y p e r e n n i a l ryegrass (Lolium perenne) are not as winter hardy as many other grasses, and were h e a v i l y damaged by the i c e encasement. New Zealand w i l d white c l o v e r ( T r i f o l i u m repens) i s f a i r l y winter hardy y e t was damaged when stored i n i c e . A l s i k e c l o v e r ( T r i f o l i u m hybridum) i s e s p e c i a l l y w e l l adapted to c o o l climates and to wet s o i l s , even t o l e r a t i n g flooded c o n d i t i o n s f o r considerable p e r i o d s . . With such c h a r a c t e r i s t i c s , i t was s u r p r i s i n g to f i n d that the p l a n t s were not only damaged by storage i n i c e , but merely by the 2-month period at -3.0°C with no i c e encasement. F o r many years, e s p e c i a l l y i n areas where i c e - s h e e t s p r e v a i l , farmers have f e l t that l i t t l e can be done about i c e - c o v e r s , except to take the consequences (50).  With a b e t t e r understanding o f the nature o f Ice-sheet  85 damage, c e r t a i n measures could be recommended that would reduce l o s s e s p a r t i c u l a r l y to forage p l a n t s . One  of the most obvious ways of coping with the problem i s by  removing or breaking the i c e - s h e e t .  A few g o l f course  superintendents  removed the i c e - c o v e r with favorable r e s u l t s during the winter of 196l/62 that badly damaged g o l f greens across the Midwest ( l 5 0 ) .  Farmers have t r i e d to break  the i c e - c r u s t with a d i s k or the lugs of a t r a c t o r , but o f t e n the weather i s so c o l d that any water from the melting broken i c e f r e e z e s back, e s p e c i a l l y at n i g h t , to form a s o l i d i c e - s h e e t again (50). The formation of i c e - s h e e t s may  o f t e n be avoided by good drainage,  e s p e c i a l l y i n areas where heavy winter r a i n s followed by f r e e z i n g can be expected.  temperatures  In areas where s l e e t or i c e storms occur, i c e - s h e e t s  w i l l form on the surface of f i e l d s , i r r e s p e c t i v e of how  w e l l they are drained.  Green keepers have n o t i c e d t h a t , f o l l o w i n g an i c e — c o v e r that l a s t e d f o r approximately 100 days, most of the damaged greens were on heavy compacted soils.  The sandier, more f r i a b l e s o i l s had l e s s damage (150). The extent of damage may be reduced by u s i n g species and v a r i e t i e s  that are able to withstand prolonged i c e - c o v e r s .  For an example, on g o l f  courses c e r t a i n bentgrasses are much more t o l e r a n t to i c e - s h e e t s than others. 'Toronto , C-15', and 1  1  'Penncross'  are among the more t o l e r a n t bentgrasses.  Ice-sheet damage i s u s u a l l y d e v a s t a t i n g on predominantly annual bluegrass greens.  Among forage p l a n t s , sweet c l o v e r and reed canarygrass have a  r e p u t a t i o n of being r e s i s t a n t to i c e - s h e e t i n j u r y . As y e t , no breeding programme has been designed s p e c i f i c a l l y to improve c e r t a i n p l a n t s i n t h e i r a b i l i t y to t o l e r a t e i c e - c o v e r s .  In recent  years breeding programmes f o r winter hardiness have made extensive use of c o l d rooms at s p e c i f i e d temperatures,  f o r r a p i d evaluations of forage crops.  86  Forage and t u r f plants could be tested f o r ice-sheet tolerance i n a s i m i l a r way,  u s i n g the boxes with temperature c o n t r o l , or by u s i n g the technique of  encasing e n t i r e pots i n i c e with the a i d of the i n s u l a t e d sand box. techniques  could a s s i s t breeders i n the development of new The use of c e r t a i n chemicals,  varieties.  incorporated i n t o the s o i l ,  capable of slowly l i b e r a t i n g 0 , i s of doubtful value. that CO^  These  and  Experiments have shown  accumulation and not 0^ d e p l e t i o n i s responsible f o r damage a s s o c i a t e d  with i c e - s h e e t s .  I f Cv, were to be l i b e r a t e d i n the s o i l , CO^  being unable to  d i f f u s e out i n t o the atmosphere, would accumulate to t o x i c l e v e l s r a p i d l y . A number of aspects of t h i s work remain to be i n v e s t i g a t e d .  The  f o l l o w i n g are some d i r e c t i o n s i n which the study could be pursued. The  rate at which CO^  accumulates and 0^ i s depleted under an i c e -  sheet i s a point of considerable i n t e r e s t , which, with the techniques a v a i l a b l e could not be determined.  The use of gas-chromatography i n s o i l atmosphere  analyses, although u s e f u l i n d e t e c t i n g and measuring 0^, CO^, gases, d i d have some disadvantages.  For one,  and a few  the gas samples could be  other with-  drawn only once from under the ice-sheet, thus, a continuous record of changes i n the composition  of the s o i l atmosphere could, not be made.  Recently,  Jensen  et a l . (6l) developed a potentiometric membrane e l e c t r o d e f o r a continuous r e c o r d i n g of the CO^ covered polarographic  i n the s o i l .  W i l l e y and Tanner (l48)  designed a membrane  electrode to permit a continuous measurement of the  concentration of the s o i l .  By u s i n g such e l e c t r o d e s the r a t e s of  0^  CO^  accumulation and 0^ d e p l e t i o n under an ice-sheet could be measured. To date, no attempt has been reported, to determine the e f f e c t of an ice-sheet i n the f i e l d , on the composition  of the s o i l atmosphere.  p a r t l y due to the sporadic occurrence of i c e - s h e e t s i n nature.  This i s  However, an  i c e - c o v e r can be produced by a p p l y i n g water to a f i e l d when the a i r temperature  87  i s w e l l below f r e e z i n g .  Such i c e - s h e e t s were formed by Beard (7)  and frequent a p p l i c a t i o n s of water to 5 by 5 f t . p l o t s . p l a n t s are most s e v e r e l y i n j u r e d by a continuous  by  light  Overwintering  forage  ice-sheet covering a l a r g e  t r a c t of land, where l a t e r a l d i f f u s i o n of gases from adjacent uncovered plays an i n s i g n i f i c a n t r o l e i n the a e r a t i o n of the ice-covered r o o t s . l a t e r a l d i f f u s i o n may stand f o r 51  soil Such  have been responsible f o r the a b i l i t y of p l a n t s to with-  days, the r a t h e r small i c e - c o v e r s produced by Beard (7).  With a  s u i t a b l e i r r i g a t i o n system l a r g e f i e l d s could be flooded during f r e e z i n g weather to  produce i c e - s h e e t s s i m i l a r to those that occur i n nature.  Both e l e c t r o d e s  and gas-sampling tubes could be used i n the f i e l d to determine the e f f e c t of the i c e - c o v e r on the s o i l atmosphere, and consequently  on the  overwintering  herbaceous p l a n t s . An a g r o - c l i m a t o l o g i c a l study on the d i s t r i b u t i o n and frequency occurrence  of  of i c e - s h e e t s , p a r t i c u l a r l y i n regions with humid microthermal  c l i m a t e s , would be d e s i r a b l e .  In t h i s way  ice-sheet covers could be d e l i n e a t e d .  areas p a r t i c u l a r l y prone to winter  Species and v a r i e t i e s r e s i s t a n t to  i c e - c o v e r s could be recommended f o r these areas, and farmers could take precautions to minimize l o s s e s . Stubble, p r o t r u d i n g through i c e , could perhaps serve as a passage f o r gas d i f f u s i o n to the r o o t s , and i n t h i s way sheet damage.  With the techniques  be of some p r o t e c t i o n against i c e -  employed i n t h i s study, passage of gases  through the stubble to and from the root system could not be detected. l a s t few years, the use of O^and C^Og been introduced (59,  In the  as t r a c e r s i n s o i l a e r a t i o n s t u d i e s has  60) and l a b e l e d 0^ has been used i n the study of i t s  t r a n s p o r t through corn r o o t s (59).  C^"  l a b e l e d CO^ and 0 ^  could be used i n  a study of gaseous d i f f u s i o n through stubble protruding through an i c e - s h e e t , and i t would be i n t e r e s t i n g to know to what extent d i f f e r e n t plant species are  88 capable of o f f e r i n g such a passage to and from t h e i r root system. The study could be pursued f u r t h e r from a plant p h y s i o l o g i c a l aspect. F o r an example, m i n i a t u r i z e d e l e c t r o d e s designed t o measure blood 0^ and CO^ l e v e l s p o s s i b l y could be adapted t o measure the concentration of the gases i n the i n t e r c e l l u l a r spaces o f the plant (45).  low 0^ l e v e l s surrounding higher  p l a n t s have been a s s o c i a t e d with the fermentation process (138).  Recently  A u b e r t i n e t a l . ( 2 ) have pointed out that there i s no s a t i s f a c t o r y c o r r e l a t i o n between the 0^ s t a t u s of the growth medium and the p l a n t ethanol content. What i s the nature o f a p l a n t ' s r e a c t i o n to changes i n the composition of the s o i l atmosphere under an i c e - s h e e t ?  What i s the p h y s i o l o g i c a l b a s i s f o r a  d i f f e r e n t i a l response to i c e - s h e e t covers? clarification.  Many s i m i l a r problems await  89  6.  SUMMARY AND CONCLUSIONS  To determine the nature o f ice-sheet damage to overwintering forage p l a n t s , a box with temperature c o n t r o l was designed and constructed. aid  o f t h i s box n a t u r a l i c e - s h e e t s were simulated i n a c o l d room.  With the  A s e r i e s of  experiments were c a r r i e d out, a t n o n i n j u r i o u s low temperatures to a s c e r t a i n the i n f l u e n c e s o f the gases i n ice^-sheet covered s o i l s .  Prom the r e s u l t s o f  the experiments the f o l l o w i n g can be concludeds (i)  Under experimental  ice-sheet covers, a t -3.0°C, carbon dioxide  accumulates i n the s o i l , i n some instances to as high as 10$, while oxygen i s depleted t o l e s s than 4$ o f the atmosphere.  Alfalfa  p l a n t s rooted i n such i c e covered s o i l s are i n j u r e d o r even k i l l e d a f t e r prolonged (ii)  periods under these c o n d i t i o n s .  An ice-sheet per se i s not damaging t o a l f a l f a , since the p l a n t s s u f f e r e d no i l l e f f e c t s when the s o i l under the i c e - c o v e r was aerated.  90 (iii)  In the present  study, approximately h a l f o f the carbon dioxide which  accumulates i n the s o i l under a r t i f i c i a l l y formed ice-sheets i s a t t r i b uted to microorganisms, while the r e s t i s probably due to root r e s p i r a t i o n . (iv)  When the carbon d i o x i d e accumulation under an ice-sheet i s a c c e l e r a t e d and brought to l e v e l s higher than would occur under n a t u r a l c o n d i t i o n s , by f l u s h i n g the s o i l with the gas, the i c e covered p l a n t s are severely damaged o r even k i l l e d  (v)  a f t e r periods' as short as 21 days.  P l a n t i n j u r y a s s o c i a t e d with ice-sheet covers a t -3.0°C, e i t h e r where the s o i l carbon d i o x i d e i s permitted artificially,  to accumulate n a t u r a l l y o r induced  appears to be p h y s i o l o g i c a l r a t h e r than mechanical.  Furthermore, carbon dioxide accumulation r a t h e r than oxygen d e p l e t i o n i s responsible f o r the i n j u r y , since- the covered p l a n t s are able t o withstand  periods up t o 3 weeks i n a- nitrogen-saturated  s o i l without  s u f f e r i n g any i l l e f f e c t s . (vi)  High l e v e l s o f both oxygen and n i t r o g e n i n i c e covered s o i l s appear to have an i n h i b i t o r y e f f e c t on root and soil-organism a c t i v i t y since far  l e s s carbon d i o x i d e was l i b e r a t e d when the s o i l was f l u s h e d with  e i t h e r of the two gases, than with carbon d i o x i d e - f r e e a i r . (vii)  A freeze-thaw-freeze  c y c l e , with moderate f r e e z i n g temperatures, and  a s s o c i a t e d with an ice-sheet does not appear t o be damaging t o alfalfa.  Continuous i c e - c o v e r s resulted<in a:greater  accumulation  "of carbon dioxide and-consequently more i n j u r y s u f f e r e d b y t h e :  „plants than where the cover was temporarily.broken ( v i i i ) Overwintering  alfalfa  by.a thaw.  p l a n t s do not absorb moisture from  water-saturated  s o i l s a t temperatures c l o s e to f r e e z i n g , and are, t h e r e f o r e , not more s u s c e p t i b l e to c o l d i n j u r y as a r e s u l t of increased t i s s u e h y d r a t i o n . (ix)  D i f f e r e n t v a r i e t i e s and species e x h i b i t e d no c l e a r - c u t c o r r e l a t i o n between r e s i s t a n c e to i c e encasement and f r o s t  hardiness.  91  BIBLIOGRAPHY  1.  ARMSTRONG, W. 1964. 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