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A critical study of the cankering of certain poplars by Cytospora chrysosperma (Pers.) Fr. with special… Bloomberg, William Joseph 1960

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A CRITICAL STUDY OF THE CANKERING OF CERTAIN POPLARS BY CYTOSPORA CHRYSOSPERMA. (PERS„) FR. WITH SPECIAL REFERENCE TO;THE WATER RELATIONS OF THE HOST William Joseph Bloomberg B.Sc.y University College of North Wales, 1943 A thesis submitted in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the Department of BIOLOGY AND BOTANY and in the Faculty of FORESTRY We accept this thesis as conforming to the standard required from candidates for the degree of Doctor of Philosophy Members of the Department of Biology and Botany Members of the Faculty of Forestry The University of British Columbia March, I960. I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o The U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r 8 , C a n a d a . Date G R A D U A T E S T U D I E S Field of Study: Forest Pathology Plant Physiology Mycology ; Forest Ecology Research in Silvics Forest Pathology Other Studies: Forest Entomology Soil Plant Relations Biometrics and Field Design . P U B L I C A T I O N S Bloomberg, W. J. 1950. Fire and spruce. For Chron. 26(2): 157-161. Bloomberg, W. J. 1953. Report on timber condtions in the Dutch Creek area. Queen's Printer, Edmonton. Bloomberg, W. J. 1956. A n e w nitric acid pulping process. Can. Pulp and Paper Ind. .9(3): 12-22. Bloomberg, W. J. 1959. Some physiological and anatomical character-istics of Populus species as related to infection by Cytospora chrysosperma (Pers.) Fr. Can. Dept. Agr. Bi-Monthly Prog. Rep. 15(2). Bloomberg, W. J. 1959. Root formation of black cottonwood cuttings in relation to region of parent shoot. For. Chron. 35(1): 13-17. .... D . J. Wort ..... R. J. Bandoni V . J. Krajina ... P. G . Haddock J. E . Bier K.. Graham J. D . Beaton J. C . Sawyer Faculty of Graduate Studies P R O G R A M M E OF T H E F I N A L O R A L E X A M I N A T I O N FOR T H E D E G R E E OF D O C T O R O F P H I L O S O P H Y of W I L L I A M JOSEPH B L O O M B E R G B.Sc, University College of North Wales, 1943 ) IN R O O M 403, E N G I N E E R I N G B U I L D I N G T U E S D A Y , A P R I L 19, 1960 A T 10:00 A . M . COMMITTEE IN CHARGE D E A N G . M . S H R U M : Chairman D . J. W O R T I. W. K E R R. J. B A N D O N I P. G . H A D D O C K K. G R A H A M J. H . G . S M I T H J. E . BIER External Examiner: DR. D O W V. B A X T E R University of Michigan, Ann Arbor, Michigan A C R I T I C A L S T U D Y O F T H E C A N K E R I N G O F C E R T A I N P O P L A R S B Y CYTOSPORA CHKYSOSPERMA (PERS.) FR. W I T H SPECIAL R E F E R E N C E T O T H E W A T E R R E L A T I O N S O F T H E H O S T . A B S T R A C T Cankering by Cytospora chrysosperma (Pers.): Fr. was investi-gated in a poplar nursery at Lulu Island, B.C. Cankers were ob-served to be more numerous in early winter than at other times and more numerous on Populus trichocarpa T . G . than on the P.x cana-densis hybrid cultivars P. 'Robusta Bachieleri' and P. 'Regenerata'. The upper part of the shoot appeared to become cankered before the lower part. Experimnts with cuttings of the three poplars showed that cankers developed from inoculations only when the bark moisture content was below a critical level. The hybrids were found to have a lower critical level than P. trichocarpa. Also, the incubation period was longer in the hybrids than in P. trichocarpa and longer in the lower part of the shoot than in the upper. Cankers were arrested when the cuttings were placed in water, the arresting time being shorter in the lower part of the shoot than in the upper part. Inocu-lated P. trichocarpa plants in the greenhouse became cankered when they were subjected to drought but the two hybrids required both drought and low relative humidity conditions for cankering to take piace. The rate of moisture loss from cuttings exposed to a uniform relative humidity was found to be faster in P. trichocarpa than in the hybrids and faster from the upper part of the shoot than from the lower part. A basis for a superior water economy in the hybrids was suggested by their anatomical characteristics which included larger pith, wider vessels, greater sieve tube zone, more numerous bark fibres, thicker periderm and fewer lenticels than in P. tricho-carpa. The lower shoot had a larger sieve tube zone, a thicker periderm and fewer lenticels than the upper part. In the nursery, the hybrids were found to have a significantly higher shoot moisture content during winter than P. trichocarpa and the lower part of the shoot had a higher moisture content than the upper part. The moisture content minima for all species oc-curred in late autumn. The results suggested that the greater resistance of the hybrids to Cytospora canker' observed in the nursery was attributable to their greater water storage capacity and to the slower rate at:which water was lost by evaporation during dormancy. The occurrence of the heaviest outbreaks of the disease in early winter could be attributed in part to the decrease in shoot moisture content in the late autumn. G R A D U A T E S T U D I E S Field of Study: Forest Pathology j Plant Physiology D. J. Wortj Mycology R. J. Banddrii Forest Ecology •. V . J. Krajina . . . . ^ Research in Silvics • P . . G , Haddock Forest Pathology J. E . Bier J Other Studies: | Forest Entomology .: K, Graham i Soil Plant Relations J. O . Beaton' Biometrics and Field Design J. C . Sawyer t P U B L I C A T I O N S f Bloomberg, W. J. 1950. Fire and spruce. For Chron. 26(2): 157-161.; Bloomberg, W. J. 1953. Report on timber condtions in the Dutch' Creek area. Queen's Printer,. Edmonton. Bloomberg, W. J. 1956. A new nitric acid pulping process. Can. Pulp and Paper Ind. 9(3): 12-22. ' | Bloomberg, W. J. 1959. Some physiological and anatomical character-istics of Populus species as related to infection by Cytospora, chrysosperma (Pers.) Fr. Can. Dept." Agr. Bi-Monthly Prog. Rep. 15(2). • Bloomberg, W. J. 1959. Root formation of black cottonwood cuttings in relation.to region of parent shoot. For. Chron. 35(1): 13-17. Faculty of Graduate Studies PROGRAMME OF THE F I N A L O R A L E X A M I N A T I O N FOR T H E D E G R E E OF D O C T O R O F P H I L O S O P H Y , ": of - W I L L I A M J O S E P H B L O O M B E R G B.Sc, University College of North Wales, 1943 IN R O O M 403,. E N G I N E E R I N G B U I L D I N G T U E S D A Y , APRIL 19, 1960 A T 10:00 A , M . COMMITTEE IN CHARGE D E A N G . M . S H R U M : Chairman D. J: W O R T J. W. K E R R. J. B A N D O N I P. G . H A D D O C K K . G R A H A M , • • J. H . G . S M I T H - J. E. BIER External Examiner: DR. D O W V. B A X T E R University of Michigan, Ann Arbor, Michigan A C R I T I C A L S T U D Y OF T H E C A N K E R I N G O F C E R T A I N P O P L A R S B Y CYTOSPORA CHRYSOSPERMA (PERS.) FR. W I T H SPECIAL R E F E R E N C E T O T H E W A T E R R E L A T I O N S O F T H E H O S T . A B S T R A C T Cankering by Cytospora chrysosperma (Pers.) Fr. was investi-gated in a poplar. nursery at Lulu Island, B.C. Cankers were ob-served to be more numerous in early winter than at other times and more numerous on Populus trichocarpa T . G . than on the P.x cana-densis hybrid cultivars P. 'Robusta Bachieleri' and P. 'Regenerata'. The upper part of the shoot appeared to become cankered before the lower part. Experimnts with cuttings of the three poplars showed that cankers developed from inoculations only when the bark moisture content was below a critical level. The hybrids were found to have a lower critical level than P. trichocarpa^ Also, the incubation period was longer in the hybrids than in P. trichocarpa and longer in the lower part of the shoot than in the upper. Cankers were arrested when the cuttings were placed in water, the arresting time being shorter in the lower part of the shoot than in the upper part. Inocu-lated P. trichocarpa plants in the greenhouse became cankered when they were subjected to drought but the two hybrids required both drought and low relative humidity conditions for cankering to take place. The rate of moisture loss from cuttings exposed to a uniform relative humidity was found to be faster in P. trichocarpa than in the hybrids and faster from the upper part of the shoot than from the lower part. A basis for a superior water economy in the hybrids was suggested by their anatomical characteristics which included larger pith, wider vessels, greater sieve tube zone, more numerous bark fibres, thicker periderm and fewer lenticels than in P. tricho-carpa. The lower shoot had a larger sieve tube zone, a thicker periderm and fewer lenticels than the upper part. In the nursery, the hybrids were found to have a significantly higher shoot moisture content during winter than P. trichocarpa and the lower part of the shoot had a higher moisture content than the upper part. The moisture content minima for all species oc-curred in late autumn. The results suggested that the greater resistance of .the; hybrids to Cytospora canker observed in the nursery was attributable to their greater water storage capacity and to the slower rate at which water was lost by evaporation during dormancy. The occurrence of the heaviest outbreaks of the disease in early winter could be attributed in part to the decrease in shoot moisture content in the late autumn. \ , . ABSTRACT Cankering by Cytospora ohrysosperma (Pers.) Fr, was investigated in a poplar nursery at Lulu Island, B. C. Cankers were observed to be more numerous in early winter than at other times and more numerous on Populus  trichocarpa T & G than on the P,x canadensis hybrid cultivars P« "Robusta Bachieleri' and P, 'Regenerata'. The upper part of the shoot appeared to become cankered before the lower part. Experiments with cuttings of the three poplars showed that cankers developed from inoculations only when the bark moisture content was below a criti c a l level. The hybrids were found to have a lower c r i t i c a l level than P. trichocarpao Also, the incubation period was longer in the hybrids than in P. trichocarpa and longer in the lower part of the shoot than in the upper. Cankers were arrested when the cuttings were placed in water, the arresting time being shorter in the lower part of the shoot than in the upper part. Inoculated P. trichocarpa plants in the greenhouse became cankered when they were subjected to drought but the two hybrids required both drought and low relative humidity conditions for cankering to take place, The rate of moisture loss from cuttings exposed to a uniform relative humidity was found to be faster in P, trichocarpa than in the hybrids and faster from the upper part of the shoot than from the lower part, A basis for a superior water economy in the hybrids was suggested by their anatom-ical characteristics which included larger pith, wider vessels, greater sieve tube zone, more numerous bark fibres, thicker periderm and fewer lenticels than in P, trichocarpa, The lower shoot had a larger sieve tube zone, a thicker periderm and fewer lenticels than the upper part. i i In the nursery, the hybrids were found to have a significantly higher shoot moisture content during winter than P. trichocarpa and the lower part of the shoot had a higher moisture content than the upper part. The moisture content minima for a l l species occurred in late autumn. The results suggested that the greater resistance of the hybrids to Cytospora canker observed in the nursery was attributable to their greater water storage capacity and to the slower rate at which water was lost by evaporation during dormancy. The occurrence of the heaviest outbreaks of the disease in early winter could be attributed in part to the decrease in shoot moisture content in the late autumn. i i i ACKNOWLEDGEMENTS The author expresses his appreciation to Dr. J.E„ Bier, Professor of Forest Pathology, for critical interest and enqouragement during this investigation., Thanks for helpful advice are also extended to Dr. Bandoni, Assistant Professor, and to Dr. G.E„ Rouse, Instructor, Department of Botany; also to Dr. J 0H 0G 0 Smith, Assistant Professor, Faculty of Forestry. The author wishes to acknowledge support of the investigation as a part of an of f i c i a l project during later stages and to express thanks for the helpful advice of Dr. R0E0 Foster, Head, Pathological Investigations, Forest Biology Laboratory, Victoria, during this period. Appreciation is expressed to West Tree Farms Ltd. for access to their plantation areas and for permission to collect specimens from them. vi TABLE OF CONTENTS Page INTRODUCTION © o o e o o o o o o o o o o o o o o o o o o o o o o o o o c e o o o o o ' t f o o o o o o o o o e o o o o o e o X LiTERAT TIRE REVIEW O O O O O O O © O O D O O e O O O Q O O O O O O O O O O O O O © e o O < O « 0 ' O « 0 O O * * O e « e 3 OBJECTIVES AND METHODS OF STUDY o © o c > o » © © o o o o o o o u © » * © i » # © o © © o o © » © * © * o 7 Experimental Material • o © t > t > ( / o o w © o o o » o o © » © o © o o © o o c o © © t > © • © © • © • © © 7 Measurement and Adjustment of Moisture Content .. ....... 8 Sample ExCXSiOXl MetllOd 000000000000000. 0600 o o . o . o o o o o o . . 0000000 10 Inoculation Procedures 0 . 0 0 0 0 0 0 . 0 0 0 0 0 0 0 o ' o - o o . o o o . o o o . o o . o . . . . . . X3 Re=isolation from Infected Material o o o o . o o o . . . . . . . « . . . . » . . . . < > < 17 ARTIFICIAL INOCULATION STUDIES o o a e o o o o o o o o o o o o o . o o o o o o o o o . o o o o o o . o 17 Effect of Leaf Formation by Poplar Cuttings on their Resistance to Cytospora Canker o . o o » o . . o o o o o . o o o o . o o o . . o o . . o o o 22 Canker Growth in Dead Cuttings 00000000000000000000000 0000.000 2^ Canker Growth in Poplar Cuttings at Different Temperatures and Moisture Contents<> Experiment 1» 00000000000000000.0000.0 25 Canker Growth in Cuttings at Different Temperatures and Moisture Contents* Experiment 2o 000000000000000000000000 28 Effect of Temperature on Growth of Cytospora in Culture 00.000 32 Effect of Relative Humidity on Canker Growth ................. 32 Critical Moisture Content for Canker Development 0..0000...000 36 Arresting of Canker Growth Following Water Uptake by Outt ill^S 00000000000000000000 o o o o o o o o o o o o o o o o o e o e o o o o o o o o e o >^0 Canker Growth in Artificially Irrigated Cuttings ............. 43 Canker Growth in Poplar Plants Subjected to Drought Ti*©&tm©ITfcS 000 0000 oo 000000000000000000000 00000000000000000000 o A 5 Summary of Inoculation Studies o o o © o » © o o « o o o o o o o o o o o o o o * o e o o o o 52 INVESTIGATIONS OF HOST CONDITIONS INFLUENCING THE RESISTANCE OF POPLARS TO CYTOSPORA' CANKER o o o o o o 000 000000000000*09000000000000000 53 Relationship Between Moisture Content of Bark and Wood ....... 53 Moisture Retention in Poplar Shoots 0000.0000.000000...0.00000 58 Anatomical Characteristics of Poplar Shoots .• 0.0....0000 63 Shoot Moisture Content of Poplars in the Field ............... 76 SUMMARY © © o * « © 0 « o » o 0 © « o o o » o o © o o « © © o o o © a o o o o © o © o o © ' » o o * o o o o © « o « o © © o o o 33 BIBLIOGRAPHY o o o o e o o o 0 0 0 0 0 0 0 0 0 0 0 o o o o o o o o o o o o o o o o o o o o o o o o o o o f f o o o o o o e 85 v i i TABLES Table 1. Table 2. Table 3. Table U* Table 5. Table 6. Table 7. Table 8. Table 9. Table 1 0 . Table 1 1 . Table 12. Table 13. Table L 4 . Table 1 5 . Comparison of weights of poplar samples saturated by water, with and without vacuum i o o e o o o u o o o o « * o o « e e o o Effect of cutting method on moisture content of Populus trichocarpa shoots at different saturation X©V0 X{3 o o o o o o o o o o o o o o o o o o o o o < i o o 0 o o o » o » * o o « o o e o o e « o o o « o o Inoculation of poplars with Cytospora chrysosperma Growth of Cytospora canker in Populus triehocarpa cuttings given different moisture treatments. Expsrimon^i X© o <> o o o © © © <> o ©«© o o o © © o © o © © <> o o o o o o <> ©«© o o © © © o o Growth of Cytospora in Populus trichocarpa cuttings given different moisture treatments. Expsrirnsnij 2o o o o o o o « © © o © o © o © © « © © o o © o o © o © © o © © * o © * © © © o e o Growth of Cytospora canker in dormant Populus  trichocarpa cuttings under different growth COIldiL't/lOXIS o o © o o o © o o o o o o e o o o o o o o o e o © « o a 9 O 0 0 » e o o e e « o e o o e ' Effect of temperature and moisture content on growth of Cytospora canker in poplar cuttings. Experiment X O O O O 9 O O O O O O O O O O 0 O O O O O O O O O O O O O O O O O O O O © O O O O O O O O O a O O O O O e -Effect of temperature and moisture content on growth of Cytospora canker in poplar cuttings. Experiment 2 O O O O O O O O O O O © O O O ( I O O © O O O O O O O O O O 0 O O O O O O O 0 9 O O O O O O e O O O O 0 O O Page 11 U 16 1921 23 27 29 Effect of temperature, relative humidity and vapour pressure on moisture loss in poplar cuttings 31 Effect of temperature on the growth of a Cytospora  chrysosperma isolate from Populus trichocarpa ......... 33 Effect of atmospheric humidity and stem moisture content on Cytospora canker growth in poplar - CTJ.'t *bXll^ S « o o e © » o o o o o o e o o o o © o o o o o o o o o o e o o o o e ' o o o o o o e o o o o o 35 Critical moisture content for infection and incubation period in poplar cuttings inoculated with Cytospora CllTyS OS J3€nD& O O O O O O O©©O0OOC©OOO0OOOO » O O O O ' O O © O O»OeoOOO0O 3^ Time lapse from immersion of Cytospora-cankered Populus trichocarpa cuttings to arrest of canker §jX*OTwLll o © « » © 0© © o © o © o o o o o * OQC0Ooooooeooooo0oooooaeo« o » « o 42 Cytospora canker growth in Populus triehocarpa cuttings given different Irrigation treatments ........ UU Infections obtained by a r t i f i c i a l Cytospora inoculation of Populus trichocarpa given different watering regimes. Experiment 1. .., > o « « « « o o « o » o o e « Q © « o o o A7 v i i i Table 16. Table 17. Table 18. Table 19. Table 20. Table 21. Table 22. Table 23. Effect of drought treatment on infection of poplar plants by Cytospora. Experiment 2<> .............. Regression of bark moisture content on wood moisture content in poplar shoots 0000000000000000 Change in moisture content of poplar cuttings exposed to room atmosphere 0000000000.0000000000001 Regression of moisture content on time in poplar cuttings exposed to room atmosphere .............. Anatomical characteristics of bark and wood in pOplXatPS e e o e o o o o o o o o o © o o o o o o o o o o o o o o o o o o o o o o o o o o o o Total vessel area in cross~sectlons of poplar SilOOiiS 00000000000000000000000000000000*0000000000 Wood moisture contents of upper and lower stem in poplar shoots 000000000000000000000000000000000 March of moisture content in poplar shoots 00000 PLATES Plate l o Natural Cytospora cankers on Populus trichocarpa cuttings occurring under storage conditions. . • Plate 2. Appearance of poplar nursery in the summer following severe winter infection of Cytospora chrysosperma. Plate 3. Relationship of root formation and Cytospora canker in 4-month old Populus trichocarpa cuttings from a plantation. Plate 4« Field inoculations of Populus spp. with Cytospora. Plate 5. Cytospora canker on Populus trichocarpa growing in greenhouse. Plate 6„ Method of taking moisture samples from one-year old Populus  trichocarpa shoots. Plate 7. Fruiting structures of Cytospora chrysosperma. Plate 8. Cytospora chrysosperma hyphae in bark of Populus trichocarpa. Plate 9. Ar t i f i c i a l inoculations of Populus trichocarpa with Cytospora. Plate 10. Growth of Cytospora canker in inoculated cuttings of Populus  trichocarpa subjected to different watering regimes. Plate 11o Growth of Cytospora canker in inoculated poplar cuttings given different moisture and temperature treatments. Plate 12. Growth rate of Cytospora on 4 per cent P.D.A. in the dark at different temperatures. Plate 13. Cytospora canker growth in poplar cuttings given different moisture and relative humidity treatments. Plate L4. Arrest of cankers in Populus trichocarpa cuttings inoculated with Cytospora. Plate 15. A r t i f i c i a l drought experiments with Populus trichocarpa. Plate 16. A r t i f i c i a l drought experiments with Populus hybrids. Plate 17. Regressions of bark moisture content on wood moisture content in six-month old Populus cuttings collected in f u l l growth. Plate 18. Regression of moisture content on time in Populus cuttings exposed to room atmosphere. Plate 19. Regression of moisture content on time in Populus cuttings exposed to room atmosphere. Plate 20. Cross-sections of the wood in six-month old poplar shoots, taken near the base of the shoot. X Plate 21. Cross-sections of the wood in six-month old poplar shoots, taken near the base of the shoot. Plate 22. Cross<=seetions of the bark in six-month old poplar shoots. Plate 23. Cross-sections of the bark in six-month old poplar shoots. Plate 24.0 Cross-sections of the periderm in six-month old poplar shoots,, taken hear the base of the shoot. INTRODUCTION During the late f a l l of 195° a heavy outbreak of Cytospora 1 N  chrvsosperma Fr. canker was observed at a poplar nursery at Lulu Island, B.C. The growing stock at the nursery comprised coppice shoots of Populus trichocarpa T & Gs which is a native of British Columbia, and four hybrid cultivars 2. £. 5Regenerata?, P. "Robusta Bachieleri' s P. "Robusta Issendorf8 and P. °Grandis?. At the time of the outbreak the shoots had completed their f i r s t growing season. In spite of the fact that the beds of each of the poplars were adjacent to each other, cankering was severe only in P. trichocarpa,, while P. "Regenerata" was lightly cankered and no cankers were observed on either P. 'Robusta' cultivars or on P. 'Grandis5. Cankers were in i t i a l l y confined to the upper region of major shootsp to lateral branches and to minor shoots. In the following January the major shoots were harvested and made into cuttings which were then stored in an unheated basement until the planting season. Further examination at the middle of March showed that approximately 20 per cent of the P. trichocarpa cuttings were heavily cankered beyond salvagej on the other hand, no cankering was observed on the other poplars. It was also noted that long sets of P. trichocarpa had much smaller and fewer cankers than the cuttings which were 6 to 10 inches long. Infection seemed to originate at any minor bark opening such 1. In the interests of brevity a l l references to Cytospora mean Cytospora  chrysosperma (Pers.) Fr. 2. Poplar nomenclature in this report follows the international code for cultivated plants. (United Nationss Food and Agriculture Organization, 1955•) Accordingly cultivar names follow directly the generic name and are placed in single quotation marks. -2-binding wounds, bark rib breakage, scuff wounds, pruning scars and even lenticels.. In some cuttings, however, similar wounds were present with-out infection suggesting that the infected individuals were in some way predisposed to the disease (Plate i)„ Periodic inspection of the nursery during the following summer failed to reveal any evidence of canker growth in the current season's shoots (Plate 2). However, in each of the two succeeding winters some cankering was again observed following defoliation but never as heavily as during the fi r s t winter and again no cankering was observed in the two suc-ceeding summers. The evidence, therefore, seemed to suggest that environ-mental conditions were involved and that these varied seasonally and from year to year. Observations over a three-year period also showed that P. trichocarpa was attacked by Melampsora occidentalis Jacks, rust while the hybTids remained free from infection. Furthermore the heaviest rust incidence occurred in the summer preceding the heaviest canker outbreak. In addition to the nursery inspection, examinations were made of the plantations in which the poplar cuttings had been set out. These revealed significant mortality from Cytospora canker only in the f i r s t year after planting. Negligible infection was observed in plants two years and older. Those cuttings suffering greatest cankering appeared to have least root development while l i t t l e cankering was found in those whose roots were well developed (Plate 3 ) . In the course of preliminary investigations no infections were obtained when two-year old Populus trichocarpa shoots growing at the University of British Columbia were inoculated with a mycelial culture of Cytospora at ten-day intervals from March to July (Plate 4). However, experiments with cuttings yielded the following results showing that under certain conditions infection could be obtained. (1) Cuttings inoculated with Cytospora. then placed in water showed very limited canker growth and this was followed by the formation of a well-defined wound periderm,, No active cankers remained subsequently. (2) Cuttings inoculated by identical procedures, but not placed in water, without exception developed active cankers which progressed until the entire cutting was cankered. (3) Cuttings which were potted in watered greenhouse soil and had put out leaves developed active cankers. In 70 per cent of the plants, however, wound periderm was formed subsequently and canker growth halted (Plate 5)« In the remainder no periderm was observed and the plants were killed by the cankers. The results obtained from the inoculations under three different growing conditions, i.e. in water, dry, and potted, illustrated the variable evidence that could be obtained when inoculation tests were carried out disregarding the host condition,. When maintained in a dry condition, a l l cuttings developed active cankers which progressed; when watered no pro-gressive cankers formed. Cuttings which were planted in soil showed an intermediate condition, that is, cankers formed but they were permanently arrested by wound periderm in most cases. In shoots growing in the field, however, no infection could be obtained at a l l . It appeared, therefore, that a more fruitful approach could be made to the problem of Cytospora canker in the nursery and plantation poplars from the standpoint of host condition, especially the moisture relations of the host. LITERATURE REVIEW Cankering of poplar and other deciduous tree: species by Cytospora has been recorded by a number of investigators in North America and Europe -A-(Longj, 1 9 1 8 ; Hubert, 1 9 2 0 $ Moss, 1 9 2 2 | S.chreiner, 1 9 3 1 j and Christensen, 1 9 4 . 0 ) , Schreiner ( 1 9 3 1 ) made an extensive review of the record of the disease and the following conclusions were drawn from this record. Certain species of poplar appeared to be more resistant than others. The attack by Cytospora was secondary to some injurious condition in the environment, e.g., drought, fire injury, or to attack by a more vigorous pathogen, Cytospora was a wound parasite. The disease was geographically widespread. Munch as early as 1 9 0 9 recognized the greater pathogenicity of this organism when wood moisture content was low. Long ( 1 9 1 8 ) correlated Cytospora infection of poplar with drought conditions. Similarly, Hubert ( 1 9 2 0 ) ascribed heavy infection to three successive dry summers. Further indirect evidence of the necessity for host predisposition to attack was contained in the report of Bier ( 1 9 3 9 ) who noted the presence of Cytospora as a secondary pathogen in Russian poplars already attacked by Septoria musiva Peek,, and by Shea-^  who found that Cytospora could attack those poplars which could be successfully inoculated with Septoria, Wright ( 1 9 5 7 ) reported that shelter belt Populus deltoides Bartr. in good soil moisture conditions showed markedly less susceptibility to Cytospora than those in poor conditions. Although no reports were found of the effect of season in Cytospora infection, several investigators have reported a striking seasonality in the resistance of poplars to infection by other canker fungi. Schmidle ( 1 9 5 3 , 1 9 5 3 A ) could only obtain significant infection by inoculations of Dothiehiza populea Sacc, et Briard in September and October and again in 3 o Shea, Keith R. Forest Pathologist, Weyerhaeuser Timber Company, Centralia, Washington, U.S.A. Personal communication to Dr. J.E. Bier. February and March. During the remainder of the year the infection rate was negligible, waterman (1957) could not obtain infection by the Dothichlza in the summer in which the inoculations were made. However, infections were observed in the following summer, i.e. after the lapse of a dormant period. On hosts other than poplar,. Van Vloten (1952) made similar observations of the seasonality of infection of Japanese larch by Phomopslg pseudotsuejae Wilson which :paly took place during the dormant season. This evidence is of interest sinee Glbbs (1957) found seasonal lows in poplar bark moisture content from November to May and lows in wood moisture content in September and March. With the exception of Munch's discovery in 1909 that a decreased water content of elm bark enhanced canker development by Nectria cinnabarina Fr., more direct evidence of a correlation between host moisture content and canker disease resis-tance has been obtained comparatively recently. Butin (1955) maintained cuttings of Populus deltoldes at various moisture contents and inoculated them with Cytospora. He reported that only cuttings which had less than a certain moisture content were infected. The crit i c a l level was between 78 to 82 per cent of the moisture content of the cutting. He also reported that a certain water deficit prevented wound healing in the cuttings thus promoting infection. Similar results (Butin, 1956) were obtained in poplar cuttings inoculated with Dothichlza populea. Bier (1959) reported that when the bark moisture content of willow cuttings was lowered to 80 per cent relative turgidity and less by drying, successful inoculation by Crytodiaporthe salicina (Gurr.) Weh. was possible. Cuttings held in water had bark moisture contents exceeding 80 per cent and did not become infected. In willow saplings, moisture content f e l l below 80 per cent during dormancy and natural cankers were observed. These were arrested with the onset of growth when the moisture content rose above 80 per cent. More recently this correlation was shown to exist in two other diseases, Fusarium canker of black cottonwood and Cephalosporium canker of western hemlock (Bier 1959A, 195?B). The depression of growth activities generally by drought has been demonstrated by a number of investigators (Addicott, 1944; Wadleigh and Gauch, I948j Helmerick and Pfeifer, 1954-1 upchurch et § 1 . , 1955j Ashton 1956). A decrease in soil moisture has been shown to cause significant reductions in photosynthesis, transpiration, respiration, leaf, stem and root elongation, root growth, flowering and other processes. These effects can be explained by references to c e l l behaviour when turgor is low, e.g. lack of expansion force in growing cells, restriction of gas exchange by closed stomates, change in enzyme activity and in ce l l permeability. Other investigators (Weimer and Barter, I921j Priestley and Woffenden, 1922) have found that wound cork formation was reduced by low tissue mois-ture content. Resin production has been found to increase at higher moisture contents (Harper, 1936). This evidence suggested that moisture plays an important role in plant disease resistance mechanisms. GauWnn (1950) believed that the plant moisture regime may effect facultative pathogens in two ways: (a) sub-optimal moisture may reduce the host's metabolic efficiency and (b) high moisture content may exclude oxygen from the tissues. With regard to the association of a heavy Cytospora canker outbreak with a severe Melampsora rust infection, Van Meiden and Van Vloten (1958) reported that in Holland Populus spp. were severely attacked by Dothlchiza populea following heavy infection with Melampsora spp. They suggested that Dothichiza entered through the leaf traces and bud scales and that the resistance of the trees to this fungus was diminished by the effect of the rust infection. OBJECTIVES AND METHODS OF STUDY The foregoing evidence from the preliminary Inoculations, from field and nursery observations and from the literature suggested the following hypotheses concerning Cytospora canker on the poplars in the nursery. (1) There was a difference in susceptibility between poplars. (2) There was a difference in susceptibility related to age or size of the shoot. (3) There was a seasonal effect in susceptibility, (4,) The mechanism of resistance was related to the water economy of the host. To test these hypotheses the following paths of investigation were indicated: a r t i f i c i a l inoculation studies correlating resistance with moisture content; a criti c a l study of the characteristics of various poplars with special reference to their moisture economies; a study of seasonal factors, especially march of moisture content and growth phase; a histological study of cell response to wounding in relation to their moisture contents; a review of the characteristics of the fungus which might elucidate the etiology in the light of the above tests. This report presents findings from the first three lines of investi-gation. Experimental Material Poplar material used throughout the study was obtained from one- and two-year old coppice shoots of the West Tree Farms Limited nursery at Lulu Island, B.C., and also from one- ahd two-year old shoots of wild poplars growing on the Endowment Lands of the University of British Columbia and at Langford, B.C. The poplars selected for investigation were P. trichocarpa. P. 'Regenerata', and P. 'Robusta Bachelieri', since these three had shown a wide range of resistance in the nursery being low, intermediate and high respectivelyo The parentage of the P. trichocarpa was not established since a number of unidentified clones were used to establish the nursery stock. The two hybrids were imported from Europe as cuttings and propagated in British Columbia. Each was stated to be a pure cultivar. For experimental purposes i t was advantageous in some cases to use cuttings taken from healthy viable shoots rather than to work with rooted plants. This procedure appeared justified since the genus Populus has the merit, frequently observed by the writer, of retaining viable functions when subdivided into lengths as small as 2 cm., e.g. root, shoot and callus for-mation. (All cuttings used in the experiments to be described were at least 10 cm. long.) Secondly, the results using cuttings were reproduced by experiments with whole plants. Thirdly, experimental tests of viability in cankered cuttings confirmed that they retained the capacity for growth and differentiationo Measurement and Adjustment of Moisture Content In many of the experiments conducted i t was necessary to determine moisture content of the plant tissues. This may be expressed on three bases: percentage of fresh weight, percentage of dry weight, and percentage of saturation. Only the latter two were used in this study. Dry weight basis is subject to variability arising from differences in bark density. While this may be serious when bark anatomy differs markedly from one sample to another, in samples from the same section of one shoot, i t should not cause a significant error. Percentage of saturation or relative tur-gidity, has the merit of eliminating the density factor from the basis and also of measuring indirectly a physiological quantity in living cells, i.e. turgor pressure. The calculation on this basis is made as follows; percentage of saturation = i n i t i a l weight of water in sample . x IQQ weight of water in the saturated sample This basis was also applied to wood moisture content in which case i t was assumed to measure the percentage of vessels containing water as op-posed to those which were empty,, This picture of the hydration of the wood was more precise than that given by the dry weight basis, and also had the aforementioned advantage of eliminating variations in density between samples. A reduction of water weight equal to 30 per cent of the dry weight removed from consideration the amount of water which x*as held in the cell 1 i walls at fibre saturation point (Brownp Panshin and Forsaith, 1952) and which was considered to be less mobile than the water in the lumen. In a number of experiments i t was necessary to adjust the percentage of saturation of a cutting to a predetermined level. This was done by taking a subsample of the cutting about 1 cm0 long, saturating i t with water, and then oven drying i t . Its weight at the required moisture content was then determined and the weight for the whole cutting at the requisite mois-ture content calculated by proportion,, Cuttings which were heavier than this weight were then dried slowly or i f too light were allowed to take up moisture until the required weight, i.e. moisture content, was reached. The most satisfactory method was to float the cuttings or samples in water at a constant temperature,. At the conclusion of each experiment the oven dry weights of the cuttings were determined as a check on calculations. In a l l cases the difference between calculated and actual weight was less than five per cent. Some possible sources of error in the saturation method of moisture measurement were evaluated. These included: (1) Biological changes during soaking. (2) Loss of solutes during soaking, thus giving a reduced dry weight. (3) Incomplete saturation of the wood sample, giving too high a moisture content. -10-The first of these was minimized by immersing the samples in distilled water at 5° G„ Loss of solutes was investigated by evaporating the im-mersion water and weighing the residue. A positive error of not exceeding three per cent, and averaging one to two per cent moisture content was foundo • Incomplete saturation of the wood sample was tested by immersion under vacuum. The differences were not found to be significant (Table l ) . Sample Excision Method In devising a technique for taking bark and wood samples consideration was given to the effect of excision method on the moisture content. Gibbs (19351 194.2) brought out the importance of water column tension within the vessels as a factor determining the moisture content of samples cut from woody stems. He reasoned that rupture of the columns would cause their rapid contraction from the cut surfaee. Thus the moisture content of a wood sample extracted from a stem by a technique which ruptured both ends of the water column simultaneously would be higher than that of a sample in which the columns had been ruptured by consecutive cuts. MaeDermott (1941) actually compared the moisture content from small sections of tree branches isolated by two simultaneous cuts with those taken by single consecutive cuts. He found significantly lower moisture contents in the samples taken by consecutive cuts and concluded that these lower values were due to withdrawal of the water columns from the sample when tension was released by cutting. Both McDermott and Gibbs found that differences due to the method of cutting were affected by transpiration conditions. Sections cut during conditions favouring rapid transpiration showed greater differences and these were presumed to be due to greater tension in the water columns. In the light of Gibbs' and McDermott's findings experiments were conducted to ascertain the importance of the excision method in the poplar material to be sampled. -11-Sample no TABLE 1 COMPARISON OF WEIGHTS OF POPLAR SAMPLES SATURATED BY WATER, WITH AND WITHOUT VACUUM Saturated weight (g) No vacuum Vacuum 18 hrs. at 15 lbs. Differ* A203 ,2.41- 2.4-7 +.06 A223 1*44 !o44 0 B i l l 1.82 1.82 0 B112 lol? 1.19 +.02 B123 1.23 1.23 0 B123 1»96 1,95 -.01 C123 3 082 3.88 + .06 C12 3.79 3.80 + .01 C113 3o62 3.62 0 C1131 4.35 4.37 + .02 A211 2.54 2.59 + .05 A21 1.90 1.95 +.05 LSD(p <.05) .17 -12-The material used consisted of Populus trichocarpa shoots approxi-mately 2 m. long. In each experiment the shoots from one stump were used. Shoots were cut off at the base and immediately transferred to the labora-tory where four sections 5 cm„ long were taken from each shoot at equi-distant points using a double-bladed hacksaw which gave two simultaneous cuts of equal depth at each stroke (Plate 6 ) . Immediately after each double cut another section was obtained adjacent to i t using only a single saw cut. A sawing device was used in preference to an instrument with shearing action in order to avoid possible displacement of water in a small sample by crushing. Precautions were taken to prevent loss of moisture by evaporation from the sections between sawing and weighing. The time between the f i r s t and second cuts did not; exceed 30 seconds. The moisture content of each section immediately after cutting was determined on a dry weight basis. Since the sections to be compared were adjacent, errors arising from differences in density were ignored. Immediately after the moisture content samples had been taken the resultant subdivided shoots were weighed and then placed in water. Periodic weighings were made until no further increase in weight was recorded, i.e. the shoots were saturated. Their i n i t i a l moisture contents were then calculated as a percentage of saturation. In the f i r s t experiment shoots were cut during dormancy at the begin-ning of March and the percentage of saturation ranged from 55 to 65. In the second experiment shoots were sampled during the growing season on July 23rd under conditions favouring rapid transpiration and the percentage of saturation was between 56 and 65. This experiment was repeated using / shoots cut one month later when transpiration conditions were also favour-able and the saturation was between 69 and 72 per cent. In the third experiment shoots were cut in August but before being sampled were allowed -13 -to take up water until they were saturated. The results, summarized in Table 2, showed that when shoots were taken during conditions favouring transpiration, the sections made by double simultaneous cuts were significantly higher in moisture content than those made by single cuts. On the other hand, when shoots from the same sample were allowed to take up water before the sections were removed, there was no significant difference between the moisture content as deter-mined by the two methods. As might be expected, the dry weight moisture contents of these saturated snoots were much higher than those in unwatered ones. In shoots which were cut in dormancy no significant differences were found between the cutting methods, even though the saturation percentage and dry weight moisture contents were lower than in either of the other experiments. In the latter case no tension was presumed to exist in the vessels since the leaves were absent. However, in order to adopt a pro-cedure which would give correct results under a l l conditions, the double-cut method was used. Inoculation Procedures For a r t i f i c i a l infections i t was desirable to devise methods which would give the greatest number of positive infections. A series of experi-ments was carried out for this purpose using two species of poplar. In the fi r s t experiment twelve cuttings 45 cm. to 50 cm. long from the youngest shoots of dormant Populus nigra trees and the same number from dormant P. trichocarpa trees were cut in February. The cuttings were free from symptoms of disease and from other abnormalities. They were brought into the laboratory and inoculated according to the following schedule and placed in an inoculation chamber. Inoculum: mycelial culture on four per cent potato dextrose agar from a single spore cluster of a Cytospora.:isolate from P. trichocarpa. TABLE 2 EFFECT OF CUTTING METHOD ON MOISTURE CONTENT OF POPULUS TRICHOCARPA SHOOTS AT DIFFERENT SATURATION LEVELS Gutting method Shoots cut July 23 Sat. % = 56 - 65 Shoots cut Shoots cut Augo 23 Aug. 23 placed in water Sato % = 69 - 72 Sate % = 100 Shoots, dormant cut March 1 Sat. % = 55 - 65 Double cut Single.cuta Difference LSD (P = .05) Average Moisture Content on Dry Weight Basis (%) 123.1 (8) 180.8 (9) 218.5 (6) 1L3.&. (8) 9.3 5.7 172.1 (9) 8.7 8.1 222.4 (6) - 3.9 4-. 4-94.5 (9) 95.5 (9) - 1.0 2.2 Figures in parenthesis show number of samples in each determination -15-Treatment: three separate inoculation methods, replicated twice on each of two cuttings, were made on each species of poplar. Treatments were as follows: ' (a) S l i t . The bark was surface sterilized by swabbing with 50 per cent methyl alcohol and then a small vertical incision was made in the stem to the depth of the cambium. The inoculum from the advancing margin of the culture was placed in the s l i t , the area of the inocu-lation wrapped loosely with mpist cotton batting and the whole wrapped in polyethylene <> (b) Scorch. A hot scalpel was applied to the bark to cause light scorching. Inoculum was applied to the surface of the scorched area and wrapped as above<> (c) Bud. The bud surface was sterilized with 50 per cent methyl alcohol, the inoculum placed on the Surface and wrapped as above. Controls were set up for each treatment. In a second experiment cuttings of P. trichocarpa and P. nigra which had been potted up in black compost greenhouse soil and whose buds had broken were inoculated with Cytospora using the. procedures (a) and (b) described above. The cuttings were kept in the laboratory next to a win-dow admitting several hours of bright light each day. The humidity in the laboratory was about 30 per cent, temperature ranged from 75° to 80° F. The plants were watered regularly. The results (Table 3) showed that either the bark scorch or the bark s l i t method was satisfactory but that bud inoculations were less effective in producing cankers. -16-TABLE 3 INOCULATION OF POPLARS WITH CYTOSPORA CHRYSOSPERMA Species trichocarpa nigra Cuttings Potted in Soil Type of inoculation S l i t Scorch Bud Total Sl i t Scorch Bud Total Total no. made 5 2 11 U 3 11 No. positive U U 0 8 A U 1 9 trichocarpa Cuttings Placed in Inoculation Chamber Sl i t 3 3 Scorch 5 U Bud U 0 Total 12 7 nigra S l i t Scorch Bud Total A U U 12 2 1 0 3 -17- . Re-isolation from Infected Material A l l isolations of the fungus from infected material were carried out using the following technique. The advance margin of the canker was located, the bark swabbed with $0 per cent alcohol and then peeled back with a sterile knife. Small fragments of cankered tissue were removed and planted on P.D.A., then incubated at 20? c. Pycnidia usually arose in culture after 10 to 12 days but orange spore tendrils were only sparsely produced in culture. Microscopic exami-nation of spores was carried out and their length, shape and colour com-pared with those given by Long (1918), Christensen (194-0), and Barnett (1955). In a l l cases they were found to be the same. Spore clusters derived from single tendrils were cultured and pycnidia again obtained in culture. After re-isolation, infected material was allowed to dry out for a period and then placed in a humidity chamber. The production of fleshy, orange spore tendrils characteristic of Cytospora chrysosperma was noted as an additional check (Plates 7 and 8). Christensen noted that the per-fect stage of Cytospora chrysosperma. i.e. Valsa sordida Nitshke was obtained in culture only with difficulty. In the experiments reported herein the perfect stage was not observed at any time in culture. ARTIFICIAL INOCULATION STUDIES The objective of the a r t i f i c i a l inoculation studies was to identify and evaluate major etiological factors in Cytospora canker of Populus  trichocarpa. P. 'Regenerataf and P. 'Robusta'. Instances have already been cited of various poplars and canker organisms in which differences in resistance were attributed to host differences, to moisture content and indirectly to seasonal effect. It was desirable to test these factors, therefore, in the relationship between the three poplars and Cytospora. In addition, the effects of temperature, relative humidity -18-and drought were also considered worthy of investigation. As a f i r s t step, the relationship of bark moisture and Cytospora canker in P. trlehoearoa cuttings was investigated. Sixteen P. trichocarpa cuttings 9 30 to 40 cm. long, a l l apparently free from natural infection, were obtained from the nursery during the dormant period. A section was taken from the mid-point of each cutting and the bark moisture content determined* Both members of the resultant pair of cuttings were then assumed to have the same value. The cuttings were inoculated with Cytospora and one member of each pair placed with the basal end in water and the ends of the other sealed with wax. The treatments were thus allotted so that comparisons could be made between them excluding genetic and other variation, among the cuttings. In addition to the inoculated cuttings, check cuttings without inoculum were set up. During the course of the experiment canker length, cutting weight and the dry weight moisture content of the bark were determined at inter-vals. For this purpose small squares of bark were removed from the uncankered portion of the stem as far as possible to avoid interference with the development of the canker. The experiment was concluded after 20 days. The results are summarized in Table 4. One week after the experiment had started the buds of a l l cuttings standing in water had broken, but no breaking had occurred in the dry treatments. Nine days after inocu-lation active cankers began to develop in the dry cuttings only and these progressed until the entire cutting was cankered or the experiment was terminated. Cytospora was re-isolated from the canker margins. Up to the end of the experiment no cankers occurred on cuttings which had been placed in water and well defined periderm was observed around the wounds in these (Plate 9). -19-TABLE 4, GROWTH OF CYTOSPORA CANKER IN POPULUS TRICHOCARPA CUTTINGS GIVEN DIFFERENT MOISTURE TREATMENTS. EXPERIMENT 1. No. of days ^ water 15 18 * 7 ^ In water 20 In D r y water Cuttings no. 3'& 4 Canker length (mm.) Bark M.C. % Wt. change % Cuttings noo 5 Canker length Bark M.C. % Wt. change % & 6 Cuttings no. 7 & 8 Canker length Bark M.C. % Wt. change % Cuttings no. 9 & 10 Canker length Bark M.C. % Wt. change % Cuttings no. 19 & 20 Canker length Bark M.C. % Wt. change % Cuttings no. 27 & 28 Canker length Bark M.C. % Wt. change % 0 177o0 0 0 162.1 0 0 172.0 0 0 158. 0 0 157.0 0 0 189.8 0 0 177.0 0 0 162. 0 0 172.0 0 0 158. 0 0 157.0 0 0 189.8 0 55.8 0 91.7 238.6 -17.3 +16.5 48.6 0 110.2 177.0 -22=6 +16.2 6.1 134.2 - 3 . 1 6.1 123.1 -24-4 7.2 U1.9 -22.4 0 265.0 +7.1 0 240.0 +10.2 0 228.2 +11.2 64.6 0 116.7 264.8 -32.2 +16.8 24.2 0 138.0 279.8 -27.8 +4.9 53.6 129.6 -28.3 35.0 120.8 -28.9 0 245.0 +10.2 +10.2 8.2 0 24.3 0 138.9 214.2 151.8 -16.8 +0.8 -21.8 +0.4 39.3 88.0 -29.1 40.0 118.4 -40.6 0 254.9 +1.0 61.8 0 100.6 251.6 -29.1 +10.9 0 197.2 +1.0 29.3 0 78.4 274.3 -37.3 -0.2 -20-Bark moisture content of each member of a pair of cuttings from the same shoot were presumed to be the same at the start. In the dry cut-tings i t f e l l continuously during the experimental period, while in the watered cuttings i t rose and then became stabilized about two weeks after the start of the experiment. Small fluctuations occurred but did not obscure the general trend. Cutting weight decreased throughout the experiment period in the dry cuttings but stayed the same or increased in the cuttings placed in water. In a second experiment the procedure was repeated using similar material and methods. Similar results were obtained. However, follow-ing the establishment of cankers some of the dry cuttings were placed in water with the result that bark moisture content rose and canker growth was halted. Upon removing the cuttings from the water the bark moisture content f e l l and canker growth resumed. By contrast, in the cuttings which had remained dry, canker growth continued unchecked while in the continuously watered cuttings no canker growth took place at a l l . The results are summarized in Table 5 and shown graphically in Plate 10. The fact that weights increased when cuttings were placed in water but decreased when they were removed from i t was taken as evidence of water uptake or loss respectively. The few cuttings which remained the same when placed in water were therefore presumed to have been at their maximum moisture content before they were placed in water. Three possible explanations were suggested by the fact that cessation of canker growth was accompanied by moisture uptake, by increase in bark moisture content and by breaking of buds. One was that low bark moisture content prevented the bark cells from making an effective defense against hyphal penetration as indicated by Butin's findings. A second was that -21-TABLE 5 GROWTH OF CYTOSPORA IN POPULUS TRICHOCARPA CUTTINGS GIVEN DIFFERENT MOISTURE TREATMENTS. EXPERIMENT 2 . Days Cutting No. 3 Treatment Bark Total Total Average moisture wt. canker increase per day content length in canker length % (mm.) (mmo) 0 Placed in water 111.0 7.8 1 it n it 12.5 7.0 7.0 2 it n n 12.3 18.2 11.2 4 It 11 " 231.6 12.5 24.9 3.3 7 n n a 305.5 12.5 24.9- 0" Cutting No. 8 0 Dry 245.8 40.6 1 Dry 171.8 33.9 2.9 2.9 2 Dry i 6 3 . 7 33.7 13.7 10.8 3 Placed in water 33.5 18.5 4.8 4 n1 it it 207.3 38.6 42 c5 24.0 5 » it n 47.5 5.6 6 II n n 50.7 3.2 7 ti tt it 218.2 40.1 50.7 0 10 Removed from water 218.1 30.2 78.6 9.2 12. n n it 117.4 19.4 16 it tt n 117.0 24.3 "144.4 6.7 Cutting No. 2 0 Dry 241.4 22.4 1 Dry 100.7 17.4 32.6 32.6 2 Dry 97.6 17.3 35.8 3.2 3 Placed in water 17.2 49.2 13.4 5 n n it 205.7 20.4 53.3 2.1 6 it it n 53.3 0 8 n tt it 181.1 25.4 53.3 0 -22-the fungus did not grow when there was too much water in its environment as Gaumann suggested. Thirdly, the formation of leaves by the cuttings produced some effect inhibitory to fungus growth* It was necessary, there-fore, to investigate these factors separately. Effect of Leaf Formation by Poplar Cuttings on their Resistance to  Cytospora Canker To test the possibility that bud breaking and leaf formation were factors in preventing canker development P. trichocarpa cuttings were made from shoots collected in mid-dormancy (January) and allotted to the following treatments; (1) Cuttings were placed in water and stored outside the laboratory window, temperature ranging from 35° to 45° F. (2) Cuttings were placed in water and stood Inside the windowsill, temperature ranging from 70° to 75° F. (3) Cuttings had ends sealed with wax and were placed in a des-iccator outside the laboratory window. (4) As in (3) but placed inside windowsill. A l l cuttings were inoculated with Cytospora. Each treatment was replicated six times. In addition, two petri plates were planted with the same inoculum and placed outside the window. Prior to treatment care was taken to keep a l l cuttings dormant by storing them outside the labor-atory. At the end of the experiment measurements were taken of bark and wood moisture content and of canker growth in a l l cuttings. For comparison, moisture contents of poplar shoots in the field were also measured. The results are shown in Table 60 Only cuttings placed in water and inside the laboratory broke their buds and formed leaves and shoots, but regardless of whether they were -23= TABLE 6 GROWTH OF CYTOSPORA CANKER IN DORMANT POPULUS TRICHOCARPA "———— ! CUTTINGS UNDER DIFFERENT GROWTH CONDITIONS Treatment Average canker length (mmo) Bark Woqd moisture moisture Dry matter basis; (%) Growth condition Dry, inside laboratory 91 81.4 38.6 Buds, not broken Dry, outside " 28 97.9 52.4 Buds not broken • In water, inside " 0 138.5 96.1 Buds broken. Several leaves and roots In water, outside " 0 129.0 179.0 Buds not broken Field trees 102.5 64.0 Dormant Growth of fungus in petri plates 55 mm. =24-inside or outside the laboratory no cuttings which were in water devel-oped cankers and a l l cuttings which were left dry did develop cankers. Cuttings in water had significantly higher bark and wood moisture con-tents than dry cuttings and shoots in the field. Canker growth was greater inside the laboratory. The finding that no cankers developed when dormant P. trichocarpa cuttings were supplied with water together with the evidence that Cytospora disease outbreaks in the field were most common in early winter suggested that the only role of dormancy in the latter situation was to reduce the moisture content of the shoots. This conclusion was supported by the fact that both bark and wood moisture contents were lower in the field shoots than in the dormant cuttings which were placed in water. Canker Growth in Dead Cuttings In seeking an alternative explanation of the infection mechanism i t was reasoned that i f the reactions of the host tissues to the fungus were eliminated and at the same time the moisture content was varied, some conclusions could be made about the role of both factors in canker development. This avenue was explored by an experiment in which 20 dormant one-year old cuttings of Populus trichocarpa were killed rapidly by being briefly plunged into boiling water. Eight inoculated and two control cuttings were stood with bases in water and the same number kept dry. After one week re-isolations were made from the bark at various distances from the inoculum and at various points on the circumference of the cutting. Bark moisture contents were also determined. Cytospora was re-isolated from a l l positions in a l l cuttings regard-less of their moisture treatment. The mean bark moisture contents of the cuttings placed in water was 234 per cent whj,le that of cuttings allowed to dry was only 31 per cent. Thus the bark moisture contents -25= of the watered dead cuttings was at least as high or higher than that of the watered viable cuttings used in other experiments, but in contrast to the latter they developed cankers. This suggested that resistance to Cytospora canker in cuttings was not due to the direct effect of water on the fungus but was exerted indirectly through the reactions of living cells. Canker Growth in Poplar Cuttings at Different Temperatures and Moisture  Contents. Experiment 1. In the previous experiments growth of Cytospora canker in Populus  trichocarpa cuttings was found to be associated with low moisture content. Since in the field P. "Robusta" and P. "Regenerata0 appeared more resist-ant than P. trichocarpa„ i t could be postulated that their resistance was attributable only to a high moisture content and that i f this were low enough they also would develop cankers. On the other hand, disproving of this hypothesis would necessarily infer some other factor in resistance which varied between the poplars. An experiment was therefore designed to test separately the effects on canker development of moisture content and the type of poplar. In addition a test of the effect of temperature was included since i t was also considered to be a possible factor in resistance either by affecting fungus growth or plant behaviour or both. To identify the effects of the factors singly and in combination, a factorial experiment was designed with fixed levels of moisture content and temperature. Nine cuttings 15 cm. long by 2 cm. diameter approxi-mately, were made from single shoots of each of the three poplars. The shoots were from 15-month old nursery stock and at the time of cutting, July 23rd, were in f u l l growth and appeared vigorous. Two treatments -a temperature and a moisture content treatment - were allotted to each cutting by random procedure. Three levels of each of these factors were chosen to cover what was considered to be a naturally occurring range. -26-The temperature treatments consisted of 0°, 10°, and 20° G. and were provided by constant temperature cabinets. Relative humidity in a l l cabinets was approximately 50 per cent. The moisture content treatments consisted of lOOj, 75, and 50 per cent of saturation. The moisture levels were obtained by the methods described previously. (See "Adjustment of Moisture Content".) The experiment was thus a factorial type consisting of 3 poplars x 3 temperature levels x 3 moisture levels. After their moisture content had been fixed, a l l cuttings were inoculated and placed in the temperature cabinets. Canker lengths and widths were measured over a ten-day period and growth expressed as mean daily growth. In addition cuttings were weighed to ascertain the changes in their moisture contents. After the experiment was concluded re-isolations of Cytospora were made from the cankers. The results (canker length only) are summar-ized in Table 7 and shown graphically in Plate 11. A l l cuttings showed some canker growth and there were no significant differences between growth either in length or width in the three forms of poplar. However, canker growth was significantly greater in length and width at 50 per cent moisture content than at 75 and 100 per cent which were not significantly different. Also canker growth in cuttings at 20° G. was significantly greater than at 10° and at 0° C. which were not significantly different. Periodic weighings showed that a l l cuttings lost moisture during the experiment. The loss was greatest at 20° C. but not significantly different between 10° and 0° C. There were no significant differences between the poplars. The results suggested that the greater resistance to Cytospora by P. 'Robusta' and P. 'Regenerata8 observed in the field was attributable to their particular moisture economies and that they were as susceptible as P. trichocarpa when their moisture contents were reduced. Thus i t TABLE 7 EFFECT OF TEMPERATURE AND MOISTURE CONTENT ON GROWTH OF CYTOSPORA CANKER IN POPLAR CUTTINGS. EXPERIMENT 1. Temperature Initial moisture content 0° C. 505? 755? 10C# 10° C. 50$ 7556- 1005? 20° C. 505?- 755? 100^ P. trichocarpa P. 'Regenerata1 P. 'Robusta' Mean daily longitudinal growth (ram.) 1.36 1.63 0.95 3.59 3.61 I.32 8.00 3.55 1.36 2.41 1.45 1.45 3.59 1.27 1.00 4.06 3.44 2.00 1.50 1.17 1.27 1.27 1.63 1.41 4.00 1.63 1.32 -28-could be inferred that in the field their moisture contents did not f a l l to the critical level for infection while that of P. trichocarpa did. While cankers grew a little; at 0° C. and in cuttings whose i n i t i a l moisture content was 100 per cent, the amount could S t i l l have been too small to be recognized as infection under field conditions in whole plants. Canker Growth in Cuttings at Different Temperatures and Moisture Contents. Experiment 2. The effects of temperature could be assumed to have at least three components: (1) Effect on hyphal growth in the tissues. (2) Effect on water loss through vapour pressure changes. (3) Effect on water loss through factors controlling the mobility of water in the plants. In order to investigate the effect on canker growth of temperature and moisture content but with moisture loss prevented, an experiment was conducted similar to the above described one with the modification of enclosing the cuttings in polyethylene bags and by means of an atomizer saturating the enclosed atmosphere with water vapour. It was found that this technique prevented loss of moisture from the cuttings as long as the temperature remained constant. As a further check cuttings were weighed at the end of the experimental period when i t was ascertained that changes of less than + 5 per cent had taken place. It was therefore considered permissible to ignore the gain or loss of water as a factor in the experiment. The results are summarized in Table 8. In contrast to the results of the fi r s t experiment, only P. trichocarpa showed significantly greater canker growth at 50 per cent moisture content. Whereas in the f i r s t TABLE 8 EFFECT OF TEMPERATURE AND MOISTURE CONTENT ON GROWTH OF CYTOSPORA CANKER IN POPLAR CUTTINGS. EXPERIMENT 2 . Temperature 0° C. 10° C. 20° C. Moisture content 50% 75%' 1005? 50% 75% 100% 50% 75% 100% Mean daily longitudinal growth (mm.) P. trichocarpa 1.45 1.05 0 2.24 0.96 0.87 11.00 1.22 0.96 P. •Regenerata' 1.20 0.94 0 0 0 0 0.99 0 0 P. 'Robusta' 0 0.93 0 0.97 0 0 0 0.68 1.42 -30-experiment some growth took place in a l l treatments,, in the seoond no measurable growth occurred in a number of cuttings* The data therefore indicated the existence of criti c a l moisture contents for canker growth and that the criti c a l level of P. trichocarpa was higher than those of the hybrids. It was surmised from the first experiment that higher temperatures had two effects on canker growth, stimulating fungus growth and increasing moisture loss. When the latter was prevented,using plastic bags, the significant increase in canker growth at higher temperatures in P. tricho-carm could be attributed to stimulation of the fungus. However, temper-ature alone had an effect only i f the moisture content f e l l below the critical level. Thus i t appeared as a secondary but independent factor in canker growth. As in the fi r s t experiment i t was reasonable to suppose that the small growth rates in the saturated P. trichocarpa cuttings and in a l l the hybrids may have been insufficient to cause a recognizable infection in the field. The effect of higher temperatures in causing greater moisture loss in the f i r s t experiment was apparently not simple. While the vapour pressure gradient at 10° C. was higher, the moisture loss was not signi-ficantly different from that at 0° C. (see Table 9). Moreover, the vapour pressure gradient at 20° C. was over 200 per cent greater than that at 0° Co but the moisture loss was only U0 per cent greater. This indicated that a limiting effect came into operation at higher temperatures. For example, water movement to the atmosphere may have attained the maximum rate made possible by the cell structure and further temperature increases produced no response. Alternatively, the higher temperature may have produced some actively retarding influence, for example by increasing the osmotic pressure of the sap. Against this, however, must be set the -31-TABLE 9 EFFECT OF TEMPERATURE, RELATIVE HUMIDITY AND VAPOUR PRESSURE ON MOISTURE LOSS IN POPLAR CUTTINGS Temp. °C. VPA(5056 R.H.) VPB(100g R.H.) VP Gradient % Moisture loss (all cuttings) 0 2.29 4.58 2.29 17.8 10 4.60 9.21 4.60 17.5 20 17.53 8.76 8.76 ' 24.4 VP^ = Vapour pressure of atmosphere in mm. Hg. VPB = Vapour pressure of moisture at barJc tissue surfaces in mm. Hg, VP Gradient = Vapour pressure gradient. -32-fact that cell permeability to water increases at higher temperatures. Effect of Temperature on Growth of Cytospora in Culture In order to obtain further information about the growth of Cytospora at different temperatures petri plates containing L, per cent potato dextrose agar medium were inoculated with equal amounts of a single mycelial culture at temperatures ranging from - 5 ° C. by five degree inter-vals to 35° C. There were four replicates at each, temperature. Cultures were maintained in the dark but were taken out daily for measurement of growth along two diameters at right angles. Results were expressed as mean daily growth (Table 10 and Plate 12). The optimum temperature for mycelial growth was approximately 25° C. No growth took place at either - 5 ° 0. or at 35° C However, when the plates were transferred to 20° C. growth immediately resumed. The optimum temperature for mycelial growth agreed with that found by Sohreiner (1931) forValsa sordida growing on poplar decoction. However, Sohreiner also found slow growth took place at 35° C» Since the fungus grew measurably at the lower temperatures and re-mained viable even below freezing point i t could presumably stay active in shoots during the winter and grow rapidly during unseasonably warm periods. On the other hand, very high summer temperatures (above 90° F.) might reduce its growth. • • t Effect of Relative Humidity on Canker Growth Two deductions which arose from the previous inoculation experiments were that low relative humidity promoted canker growth and that the hybrids had a lower criti c a l moisture oontent for canker growth * than P. trichocarpa• The; f i r s t wasuites.ted by an experiment, in which= inoculated: cuttings of the three poplars were exposed to different relative humidities. For -33-TABLE 10 EFFECT OF TEMPERATURE ON THE GROWTH OF A CYTOSPORA  CHRYSOSPERMA ISOLATE FROM POPULUS TRICHOCARPA V Temperature Average diameter growth per day (lmrio) - 5 0 0 2.0 5 4.3 10 4.9 15 7.7 20 12.9 25 16.7 30 8.7 35 0 Growth medium was 4 per cent potato dextrose agar. this purpose j, cuttings 25 cm long and 2 .5 cm in diameter were made from single shoots of vigorous four-month old P. trichocarpa0 P. "Regenerata" and P. 'Robusta' which were cut in August. Nine cuttings of each poplar were made, using only the basal quarter of each shoot to minimize variation. Treatments were allotted to the cuttings so that for each type of poplar there were cuttings with three levels of moisture content - 5 0 , 7 5 , and 100 per cent of saturation respectively - exposed to three levels of relative humidity - approximately 100, 6 5 , and 50 per cent. The relative humidity levels were obtained by drawing a stream of air through approp-riate saturated solutions of salts and thence through a desiccator con-taining the cuttings. The relative humidity within each desiccator was checked by color indicator paper. The treatments were carried out at room temperature (approximately 70° F.). A l l exposed wood surfaces on the cuttings were thoroughly sealed with wax and the cuttings inoculated. Periodically canker lengths were measured and cuttings were weighed to determine moisture loss. The experiment period was 20 days. Results were expressed as mean daily canker growth (Table 11) and canker growth in P. trichocarpa was significantly greater than in the hybrids (Plate 13). Growth was significantly less in cuttings atHOOiper cent than in those at 50 and 65 per cent R.H. The latter were not sig-nificantly different from each other. Growth was significantly greater in cuttings whose moisture content was 50 per cent than in those with 75 and 100 per cent. The latter were not significantly different from each other. Cutting moisture contents at the end of the experiment were lowest in the 50 per cent R.H. treatment, intermediate at 65 and highest at 100 per cent R.H., a l l differences being significant. There were, however, no significant differences between the three poplars. The fact that canker growth was greatest in cuttings exposed to, 50 TABLE 11 EFFECT OF ATMOSPHERIC"HUMIDITY AND STEM MOISTURE CONTENT ON CYTOSPORA CANKER GROWTH "IN POPLAR CUTTINGS RH-5O56 RH 65$ _ RH 100$ Moisture content 50$ 75$ 100$ 50$ 75$ 100$ 50$ 75$ 100$ (Mean daily longitudinal canker growth mm.) P. trichocarpa 10.33 •7.77 1.00 15.3 7.2 1.0 7.22 1.00 1.22 P. 'Regenerata' 10.92 3.11 1.50 5.0 1.6 1.7 1.33 1.23 1.00 P. 'Robusta' 11.58 0.80 1.00 6.5 1.0 1.0 I.45 1.00 1.00 -36= per cent R.H. can be explained by the greater desiccation which was indicated by their greater weight loss. The greater growth in cuttings with a 50 per cent moisture content agreed with the results of the previous experiments in which this factor was tested and the greater canker growth in P. trichocarpa could be explained in terms of its higher criti c a l moisture content for canker development. The reason for the lack of dif-ference between the growth at 65 per cent and 50 per cent R.H. was not explainable. If the aboye findings were applicable to field conditions then i t would follow that a n7 conditions causing low relative humidity would promote canker outbreaks. A parallel conclusion was reached by Mooi (1948) that dry frosty winds caused greater susceptibility of willow to Cryptodiaporthe canker. To summarize the conclusions regarding optimum etiological conditions for Cytospora canker, temperature had a directly proportional effect on canker growth by stimulation of the fungus and by increasing, up to a point, the moisture loss from shoots^while low relative humidity promoted canker growth presumably by desiccating the tissues. Critical Moisture Content for Canker Development In the three inoculation experiments just described, some evidence was obtained that the c r i t i c a l stem moisture content for Cytospora canker development was different in Populus trichocarpa from that of the hybrids. As a more comprehensive test of this, an experiment was designed to ascer-tain not only differences between poplars, but also between shoots taken from different stumps and between different regions of the shoot. To determine whether there was an association between Melampsora rust infection and Cytospora canker development, as suggested by the nursery observations, shoots of both heavily rusted and lightly rusted P. trichocarpa were in-cluded for comparison. The basis for differentiating the degrees of rust -37-infection was by a visual estimate of rusting and defoliation.. Also, as a test of differences due to the growing conditions, shoots from wild poplars growing on the University Endowment Lands were used* Single shoots from three different stumps in each poplar were cut from the nursery in October just prior to leaf fallo Each shoot was then divided into sections of equal length and two cuttings 30 cm<. long were made from both the basal quarter and from the second quarter from the tip. The cuttings were brought to saturation, a l l their exposed wood surfaces sealed with wax and they were inoculated with Cytospora. The cuttings were placed in small wooden stands on the laboratory-bench and exposed to room atmosphere (approximately 70° F., 50 per cent R.H.). They were examined daily for canker development. As soon as canker appeared, infection was considered to have taken place and the bark, wood and total stem moisture contents of the cuttings were deter-mined from a subsample. These were designated "critical moisture contents for infection" or "critical moisture contents". The results (Table 12) were based on 55 out of the 60 cuttings which developed cankers, five results being obscured by natural contamination. The hybrids had significantly lower c r i t i c a l stem moisture contents than any of the P. trichocarpa groups. However, the two hybrids did not differ significantly from each other, nor did the three P. trichocarpa groups differ significantly. There were no differences between the shoot regions except in the wild P. trichocarpa which had a signifi-cantly higher criti c a l moisture content in the upper shoot than the lower. Stumps within poplars showed significant differences in their critical stem moisture contents. There were no significant differences between poplars in the critical moisture content of the wood alone, nor did the upper part of the shoot -38-TABLE 12 CRITICAL MOISTURE CONTENT FOR INFECTION AND.INCUBATION PERIOD IN, POPLAR CUTTINGS INOCULATED WITH CYTOSPORA CHRYSOSPERMA Species Quarter Moisture content at infection Bark Wood Stem (per cent saturation) Incubation period (days) P. trichocarpa 2 4 68.0 74.1 23.5 19.1 43.9 37.0 18 28 mean 71.0 21.3 40.4 23 P. trichocarpa (rusted) 2 4 mean 65.8 75.6 70.7 26.3 28.1 27.2 44.8 42.2 43.5 19 23 21 P. trichocarpa Twildl 2 4 mean 62.8 71.5. 67.2 24.6 13.3 18.9 46.9 28.1 37.5 13 24 P.. 'Regenerata' 2 4 mean 57.2 62.7 59.9 24.3 22.4 23.3 26.6 30.8 28.9 32 47 39 P. 'Robusta' 2 4 51.4 54.9 17.5 19.5 23.3 26.0 33 43 mean 53.2 18.5 24.7 38 Quarter 2 = second quarter from tip. Quarter 4 = basal quarter. , -3.9= differ from the lower except in the wild P. trichocarpa in which the upper shoot region had a higher cr i t i c a l moisture content than the lower. There were sighTficant differences between stumps. There were significant differences between poplars in the criti c a l moisture content of the bark alone. P. 'Robusta' had the lowest value, P. 'Regenerata' was intermediate and P. trichocarpa had the highest value. There were no significant differences between members of the P. tricho-carpa group. The cr i t i c a l bark moisture content of the upper shoot was significantly lower than that of the lower shoot. Differences between stumps were not significant. Measurement of the time required to produce infection, i.e. incu-bation period, showed that It took significantly longer for cankers to form on cuttings of P. "Robusta" and P. "Regenerata? than on P. tricho-carpa. but the three groups of the latter species did not differ from each other (Table 12). The incubation periods in the cuttings from the upper shoot were significantly shorter than in those from the lower shoot except in the heavily rusted P. trichocarpa in which they were the same. The differences between poplars in c r i t i c a l moisture content of the total stem (bark plus wood) found in the previous experiments could thus be attributed to the lower critical bark moisture content of the hybrids since their wood moisture contents at infection were the same. At the same drying rate, therefore, the criti c a l moisture content would be reached sooner in P. trichocarpa. Moreover, differences were large, approaching 20 per cent between P. 'Robusta' and P. trichocarpa. This could account at least in part for the shorter incubation period in the latter species. On the other hand, criti c a l values did not differ between the lower and upper .shoot but incubation periods were shorter in the latter. This could only be explained by a more rapid drying rate in the upper shoot. -40-It was significant that in the nursery P. "Robusta", which had the lowest critic a l bark value, did not become cankered while P. trichocarpa0 with the highest, was the most heavily cankered. P. "Regenerate5 was inter-mediate in both critical bark moisture and degree of cankering. Since there were no differences in either incubation period or cr i t i c a l moisture content between heavily rusted and lightly rusted P. triehocarpa. the results threw no more light on the association of Cytospora canker and rust infection. The lower criti c a l bark moisture content in the upper shoot was not easily explainable. Actual differences were relatively small, being less than 10 per cent, and may have been due to the greater physiological activity of the cells in the younger part of the shoot thus enabling them to resist the fungus even though their moisture contents were lower than the cells of the older, lower shoot. The critical moisture content of the shoots in the three poplars ranging from 24 to 40 per cent contrasted with But in's (1955) values for Populus deltoides ranging from 78 to 82 per cent. As far as could be deter-mined from his paper, Butin collected his cuttings in early winter (October 1954). If the moisture content in this species was at the annual minimum, as Gibbs (1957) found in Eastern Canada, then the resultant c r i t i c a l moisture content for canker development may have been similar to that of the three nursery poplars. Arresting of Canker Growth Following Water Uptake by Cuttings Another aspect of Cytospora infection of poplars was the arresting of canker growth and the question of how this varied. Some evidence on this point was provided by the i n i t i a l experiments in that cankers were arrested when cuttings were placed in water. To examine this aspect more - a -critically the 36 Populus trichocarpa cuttings from the preceding experiment were placed with their bases in water and enclosed in plastic bags immediately after the formation of a canker, the object being to allow uptake of water and to prevent evaporation (Plate 14)« Cankers were measured daily in length and width. Arresting of a canker was defined as having occurred when two successive measurements were the same. Results are shown in Table 13. Excluding three cuttings in which con-tamination developed, canker growth was arrested in a l l cuttings placed in water. Cankers on the cuttings from the lower shoot were arrested more quickly than on those from the upper, average times being 6.0 days and 15.5 days re-spectively. There were no significant differences between wild, lightly rusted and heavily rusted groups nor were significant differences found be-tween stumps. Twenty-eight cuttings produced at least one and averaged five adventitious roots. The arresting of cankers when cuttings were placed in water showed that the moisture mechanism in canker growth was reversible. The earlier arresting of cankers In cuttings from the lower shoot may have been due to a more rapid rehydration of wood and bark tissues following immersion. In the field, the supply of available moisture and the absorptive ability of the root might be criti c a l factors in the recovery of cankered trees. If both conditions were optimal, canker growth would be arrested, and in the lower shoots before the upper. Regardless of the water supply however, i f the roots were inactive the water balance of the tree would not be restored and canker growth would continue. Following lengthy droughts, the roots might become suberized and Inactive and re-activation might not be immediate when water again became available. The latter circumstances might account for canker outbreaks during the winter follow-ing a very dry summer. =42-TABLE 13 TIME LAPSE FROM IMMERSION OF CXTOSPORA-CANKERED POPULUS TRICHOCARPA CUTTINGS TO .ARREST OF CANKER GROWTH Species Quarter Days elapsed P. trichocarpa 2 12.7 " 4 6.5 " mean 9.6 P. trichocarpa (rusted) 2 10.5 « 4 7.2 " mean 8.9 P. trichocarpa (wild) 2 17.5 » • 4 4.4 " mean 11.0 Grand average it n 2 4 13.5 6.0 -43-Canker Growth In Artificially Irrigated Cuttings To examine the effects of a variable moisture supply on,canker development as opposed to the fixed levels used In the preceding experi-ments , a method was devised for ar t i f i c i a l l y irrigating poplar cuttings. Eight cuttings of Populus trichocarpa 20 cm. long and of approximately 2 cm. diameter were made from several shoots of the same stump. In each cutting, the morphological base was prepared by peeling a ring of bark from the wood and fitting the end Into a rubber tube. The cuttings were then inoculated with Cytospora. Water was supplied to each cutting from a burette, the nozzle of which fitted into the other end of the rubber tube. The flow was regulated by opening and closing the burette tap, and the consumption of water by each cutting was read from the graduated cylinder. Water levels in these cylinders were adjusted daily to maintain a constant head. Four cuttings were permitted to draw an unrestricted water supply. In four others the supply was restricted by closing a tap for various periods from one to four days, but with f u l l supply between restrictions. At the start of the experiment, a l l cuttings were given five days* un-interrupted water supply. The duration of the experiment was one month. The laboratory atmosphere averaged 70° F. and 40 per cent R. H. through-out the experimental period. Despite the fact that they were inverted, the cuttings showed no abnormal effects. Breaking of buds and development of shoots took place throughout the duration of the experiment. The results are shown in Table 14. Cankers formed on a l l cuttings whose water supply was restricted, whereas cuttings which received un-restricted water did not form cankers. At the end of the experiment bark moisture contents of cankered cuttings were significantly lower than uncankered. - A C -TABLE 14 GYTOSPORA CANKER GROWTH IN POPULUS TRICHOCARPA CUTTINGS GIVEN DIFFERENT IRRIGATION TREATMENTS Cutting no. No. days water supplied No. days water withheld Total water supplied (oo.) Longitudinal canker growth (mm.) Bark moisture content ($ dry wt.) 1 20 11 36.2 5 179 3 20 11 4 0 c 9 20 178 5 20 11 43 c 8 1 4 1 5 9 2 31 0 5Q.1 0 200 4 31 0 48.0 0 198 6 31 0 57 o3 0 178 -45-A second experiment was carried out using funnels instead of burettes and without actual measurement of the water supplied to the cuttings. Regulation of the water supply in restricted treatments was by a pinch clamp. The results were the same as in the fi r s t experiment - four cut-tings which had their water supply interrupted developed cankers, whereas five cuttings whose water supply was unrestricted did not develop cankers. Since the experiments were carried out in a warm laboratory atmosphere with low relative humidities, loss of moisture by evaporation from the lenticels and from the developing shoots was probably appreciable. The higher bark moisture contents in the fully irrigated cuttings indicated that the loss by evaporation had been replaced. The cuttings given re-stricted watering, however, had lower bark moisture contents indicating that the loss had not been balanced by intake. By analogy infections in the field could be attributed to the combined effects of restricted water absorption by roots and water loss by evaporation and transpiration. Canker Growth in Poplar Plants Subjected to Drought Treatments Cytospora canker in growing plants was investigated by subjecting inoculated plants to high and low watering regimes in the greenhouse. Two series of experiments were conducted, one using Populus trichocarpa and one using P. 'Regenerate' and P. 'Robusta' together. In the f i r s t , six P. trichocarpa saplings were grown in 10-inch crocks from cuttings taken from the same parent stump. At the time of inoculation they were five months old and similar in size and development. They were approxi-mately 1 m. t a l l , with well developed foliage and appeared vigorous. Prior to the start of the experiment a l l plants were watered daily. The rooting medium was a sandy loam without any amendments. The experiment was conducted from June to September. Three of the saplings were randomly chosen and designated for drought treatment and the remaining three for controls. The treatment consisted of withholding water until wilting occurred in the leaves and petioles and then bringing the soil up to field capacity. This procedure was repeated for the duration of the experiment. The control plants received daily watering. At the commencement of the treatments a l l plants were inoculated with Cytospora. two inoculations being made on each shoot, and one check wound without inoculum made above these. Twelve days after inoculation, pronounced periderm formed in a l l plants at the wound and no canker was observed. One month after the original in-oculations, a second series of inoculations were made and three weeks later definite narrow cankers at the margins of the wounds occurred in the drought-ed plants, but not in the controls. Although there was no advance of the canker, no growth periderm formation was visible in the droughted plantsj in the watered plants, however, relatively large callus formation was ob-served. Following further treatments the droughted plants defoliated, but 10 to 15 days later put out new foliage. This consisted of fewer and smaller leaves than those of the controls. A third series of inoculations was then carried out. Following this, well defined cankers developed in the droughted plants. As drought treat-ments continued, the cankers advanced slowly until they covered the entire stems. Cytospora was re-isolated from the margins. No cankers had occurred in any of the controls at the time the experiment was discontinued. Results are summarized in Table 15. Before the cankers had advanced into the root systems, a test for root pressure was made. The stem of each plant was cut off one inch above ground level and a rubber tube fitted tightly over the stump. This was connected to a small bore glass tube 80 cm. long which was supported in a vertical position. The root system was then steeped in water. In a l l the -47-TABLE 1 5 INFECTIONS OBTAINED BY ARTIFICIAL CYTOSPORA INOCULATION OF POPULUS  TRICHOCARPA GIVEN DIFFERENT WATERING REGIMES. EXPERIMENT 1. Tree no. Treatment Inoculation series 1st 2nd 3rd Lesion length (mm.) 2 weeks after inoculation i 1 Drought 0 + 12.5 2 Drought 0 + 23.0 3 Drought 0 + 19.0 A Watered 0 0 0 5 Watered 0 0 0 6 Watered 0 0 0 + Latent lesion control plants water exuded from the stump and rose to the top of the glass tube but no sap rise occurred in the droughted plants. As a final investi-gation the root systems of a l l plants were washed free of soil and their development compared. Plants which had been subjected to drought treatment had a volume of roots approximately one-half that of the watered plants (Plate 15). To summarize the results, droughted P. trichocarpa plants developed Cytospora cankers after a number of cycles of treatment. During the treatment plants defoliated but subsequently put out new foliage. The root systems of treated plants were smaller than those of the controls and produced no exudation when steeped in water. In the control plants by contrast, no cankers developed, no defoliation occurred, and exudation took place from the roots steeped in water. In the second experiment the poplar material was five-month old shoots of P. 'Regenerata' and P. 'Robusta' grown from cuttings. They were planted in sandy loam soil in 12-inch pots. When they were two months old and appeared to be well established, 17 plants were allocated randomly to a drought treatment and 18 kept for controls. Oats were sown in the soil of the pots to be droughted in order to provide a uniform depletion of soil moisture by rooting in a l l regions of the pot. Treatment consisted of withholding watering until the leaves and petioles became flaccid and then bringing the soil up to field capacity. This process was re-peated for the duration of the experiment. Controls were watered daily and no oats were sown in their pots. Controls and treated plants were inoculated with Cytospora. One inoculation was made on each shoot but where a cutting bore two shoots, check inoculation without inoculum was made on the second shoot. The inoculations were repeated after one month. Plants were maintained in -4-9- , the normal greenhouse environment during June through September. During this period the droughted plants had been wilted four times. In mid-October they began to defoliate prematurely and entered dormancy evidently due to drought conditions. The watered plants did not defoliate. During this period of treatment no infections were obtained. Periderm formation was pronounced in watered plants and to a l l appearances the infections were completely healed. In droughted plants, however, periderm formation was much less prolific and the fungus was observed to establish on the wound area. As a second phase of the treatment, one-half the droughted plants and one-half the controls were transferred from the greenhouse to the laboratory where the same treatment was continued. The remainder of the plants were kept in the greenhouse. Temperature and light conditions were approxi-mately similar in both locations but the laboratory had a mean relative humidity of 50 per cent contrasted with a mean of 93 per cent in the green-house, the ranges being 48 to 54 per cent and 65 to 100 per cent respect-ively. Results are shown in Table 16. Cankers developed in four out of the five P. 'Regenerate' and two out of the three P. 'Robusta' which were given drought treatment in the laboratory. Cankers developed in only one out of the five P. 'Regenerata' and none out of the three P. 'Robusta' which were given drought treatment in the greenhouse. No cankers developed in watered plants either in the laboratory or the greenhouse. A l l cankers progressed until the shoots were killed. Cytospora was recovered from the canker margins. As soon as cankers had formed, bark samples were taken from the shoots of a l l poplars and the moisture content determined. Soil moisture content was determined in a l l pots. Droughted poplars in the laboratory had the TABLE 16 EFFECT OF DROUGHT TREATMENT ON INFECTION OF POPLAR PLANTS BY CYTOSPORA. EXPERIMENT 2. Greenhouse (R.H. 92.5$) Watered P. 'Regenerata' 83.8 P. 'Robusta' 76.2 Both species 80.0 Soil moisture content $ 35.0 0° 5 0 5 _0 10 Droughted B.M.C.a Infec- B.M.C, . $ tions . $ 72.1 62.4 68.6 Infec* tions 1 5 0 3 1 8 Laboratory (R.H. 50$) Watered B.M.C. Ihfec= -$• tions 72.7 65.8 70.1 0 5 0 3 0 8 Droughted B.M.C 42.7 38.5 41.2 Infec-tions 4 6 2 3 6 9 9.9 35.9 8.8 a B.M.C. = Bark moisture content. D Numerator refers to number of successful inoculations, denominator refers to total number inoculated. ~51= lowest bark moisture content while watered plants in the greenhouse had the highest. Watered poplars in the laboratory and droxighted poplars in the greenhouse had approximately the same bark moisture contents. P. •Regenerata" had higher bark moisture contents than P. "Robusta". In a l l pots droughted soil had a lower moisture content than watered, but there was no difference caused by location. Since droughted plants defoliated prematurely presumably owing to drought-induced dormancy, their viability was established by main-taining them in the greenhouse until after five months they broke and resumed vigorous growth (Plate 16). P. "Robusta' and P. "Regenerata" bark moisture contents varied directly as both atmospheric moisture and soil moisture, but only when the lower levels of each factor were in combination was the bark apparently desiccated sufficiently to result in cankering. In P. trichocarpa. on the other hand, drought treatments produced cankers at the higher greenhouse relative humidity. These results agreed with the results of similar experiments with cuttings in that only a combination of low humidity and low stem moisture content produced infection in the hybrids while only a low stem moisture content was necessary for the infection of P. tricho-carpa . The bark of the former, therefore, appeared to reach the critical moisture content for infection only when drying conditions were severe. In this respect i t was interesting to compare European reports (Muller-Stoll, 1950) of slight to moderate Cytospora infection in P. 'Robusta' with its freedom from the disease in the Vancouver: area where humidity is; generally high, soil moisture abundant, and the growing season rela-tively long. The differences in periderm production between droughted and un-droughted plants could be attributable to the reduction of cel l division -52-and turgor In the callus tissues of the droughted plants. Vigorous periderm production healed the inoculation,, but poorly developed periderm allowed latent cankers to become established and subsequently these cankers spread beyond the margin of the inoculation wound. Summary of Inoculation Studies To summarize the findings of the inoculation studies, a positive correlation was found between the resistance of poplars to Cytospora canker and the moisture content of the host, bark moisture content appearing to be the important factor. Field observations that the hybrids were more resistant than P. trichocarpa agreed with the experi-mental findings that a lower cr i t i c a l moisture content and a longer incubation period were required for infection of the hybrids. Similarly, the greater susceptibility of the upper part of the shoot as observed in the field could be attributed to its shorter incubation period. With-in the temperature range 0° to 20° C , Cytospora canker growth in cut-tings varied as the temperature. This effect was presumed to have at least three components which also varied as the temperaturet. fungus growth, evaporation from the lenticels and water mobility in the tissues. That the fungus growth did increase with temperature was shown by culturing experiments. Low relative humidity was found to cause significantly greater canker growth and appeared to operate by reducing the moisture content of the shoot. The moisture mechanism in canker growth appeared to be reversible and cankers were arrested when the shoot was placed in water. They were arrested more rapidly In the lower shoot than in the upper. High moisture content did not appear to act directly in reducing the growth of the canker but rather through its effect on the host. Apart from its possible -53-effect on the moisture mechanism the dormancy of the host had no effect on resistance,. The general conclusion,, therefore, was that the more efficient water economy of the hybrid poplars and of the lower part of the shoot maintained the tissues above the criti c a l level for infection. Ecological factors which produced a lower availability of soil moisture and desic-cating atmospheric conditions would appear to favour infection. INVESTIGATIONS OF HOST CONDITIONS INFLUENCING THE RESISTANCE OF POPLARS TO CYTOSPORA CANKER In order to seek a comprehensive explanation of the findings of the inoculation studies i t appeared necessary to ascertain, f i r s t l y , the relationship of bark moisture content to wood moisture content in the three poplars; secondly, the differences, i f any, between poplars and between the upper and lower shoot in ability- to retain moisture under desiccating conditions, and finally the differences, i f any, in moisture content between poplars and between shoot regions which occurred under field conditions. Relationship Between Moisture Content of Bark and Wood Lateral movement of water in the wood of various plants has been found to take place readily (Auchter, 1923) and the vascular rays have been suggested as the probable path (Meyer and Anderson, 1952). It has been presumed that water moves from the xylem to the neighbouring cells by diffusion (Crafts, Currier and Stocking, 1949) and by ar t i f i c i a l l y raising and lowering osmotic pressure in the wood of willow shoots corresponding decreases and increases of the turgor of the sieve tubes were obtained (Weatherby et a l , 1959). Thus, from the above evidence wood may be likened to a water reservoir for the bark and the hypothesis put forward that bark moisture content is determined to some extent at least by wood moisture content, io6o that the former shows a significant regression on the latter. In order to test this hypothesis, the following experiment was con-ducted . A single maj or shoot of each of the three poplars was cut in the nursery and the basal half divided into nine sections of equal length, the shoots were in f u l l growth (August) and were between 2 cm. and 3 cm. in diameter. By the technique described under "Methods" the wood moisture contents of the sections were adjusted to cover a range from approximately 10 to 100 per cent saturation. Two bark samples were then removed from each section with a No. A cork borer and their moisture contents determined. The regressions of bark moisture content and wood moisture content were calculated and are shown in Table 17 and in Plate 17. Individual regressions of a l l three poplars were highly, significant (P < .01). Also the regressions of P. trichocarpa and P. 'Robusta' were significantly different In both slope and mean. Differences between these poplars and P. 'Regenerate8 were not significant. The regressions, therefore, supported the hypothesis that wood moisture content exerted a directly proportional effect on bark moisture content and that the regres-sion effect was not the same In a l l poplars, indicating possibly a differ-ence in bark and wood anatomies. Since bark/wood moisture content regression was demonstrated in excised shoot sections i t was considered desirable to confirm its exist-ence in a living tree under field conditions. For this purpose, and in order to obtain a sufficiently wide range of moisture contents, wood and bark samples were taken at 8s00 a.m. and at 3s00 p.m. on four separate days during a prolonged summer drought from a four-year old P. trichocarpa tree growing at Langford, B. C. The samples were extracted from the tree -55-TABLE 17 REGRESSION OF BARK MOISTURE CONTENT ON WOOD MOISTURE CONTENT IN POPLAR SHOOTS Species Regression P. trichocarpa Y = 5 3 . 9 + . 4 5 X P. 'Regenerata' Y = + . 5 9 X P. 'Robusta' Y = 4 6 . 0 + . 5 0 X - 5 6 -wlth a No. 5 cork borer and consisted of a disc of bark with about 5 mmo thickness of wood attached to i t . The bark was then detached from the wood in the laboratory and the Individual moisture contents determined. Four samples were taken in the morning and four in the afternoon. Each sample consisted of two sub-samples taken adjacent In the bark. Wood moisture contents obtained by this sampling method ranged from 3 8 . 3 to 6 3 . 0 per cent. Bark moisture ranged from 82,0 to 9 2 . 7 per cent. The regression of bark moisture content on wood moisture content was highly significant and compared with that of the excised shoots as follows: Excised shoots U5*U + -59X Trees 6 0 . 7 + .54X Significant differences were found between the adjusted means, but not between the slopes. The findings from the tree samples, therefore, strengthened the evidence that wood moisture content had a directly proportional effect on bark moisture. The similarity of the slope effect in four-month old nursery shoots and in a four-year old wild tree also suggested that this may have been the characteristic of the species. The difference in means of the regressions could reasonably be attributed to other differ-ences such as anatomy. A general theoretical explanation for the significance of bark/wood i moisture regression could be put forward along the following lines. If moisture is lost from the bark via the lenticels then the diffusion pressure deficit of the outer cortex cells should Increase and an osmotic gradient would be established via the rays into the wood. The bark cells would maintain f u l l turgidlty only i f the rate of water entering by diffusion from the wood equalled the rate of loss by evaporation. MacDougall. (193$), MacDermott (1941), and Gibbs (1957) have a l l found evidence of tension in the wood of trees while rapid transpiration was taking place. If such tension in the vessels exceeded the D.P.D. of the bark cells no water would move from the wood to the bark, and i f the bark continued to lose moisture by evaporation to the atmosphere, i t would become drier. On the other hand, i f the tension in the vessels was less than the bark D.P.D., then water would move from the wood to the bark at a rate proportional to the difference in their respective pressures. A corresponding increase in the bark turgor could also be anticipated. Tension in the vessels during the growing season would obviously depend on the difference in rates of transpiration and moisture uptake by the roots. During summer droughts, then, bark moisture content is likely to be low. In dormancy, water loss by deciduous trees can only take place via the bark while water uptake, in some species including Populus. results from root pressure. If, during the winter, accumulated moisture losses exceed the moisture supplied by root pressure, then the wood and consequently the bark would become drier. Thus while the in-oculation experiments showed that canker growth was inversely correlated with bark moisture content, the regressions indicated that wood moisture was the basic factor. Therefore, in poplars growing under the same field conditions, a shoot which had a higher wood moisture content than another would be less likely to be infected with Cytospora provided (a) their critical bark moisture contents for infection were the same and (b) their rates of moisture loss were equal. The inoculation experiments showed that P. trichocarpa had, in fact, a higher critical moisture content. Thus i f the rate of moisture loss In this species was not less than that of the hybrids then i t would become infected sooner. -58-Moistttre Retention In Poplar Shoots In order to compare the rates of moisture loss from shoots an experiment was conducted as follows . For each poplar 3 stumps were randomly chosen and from each stump a single shoot was cut, also by random procedure. This was done in the late f a l l just prior to defol-iation and the shoots were, therefore, one growing season old and had formed terminal buds. In order to investigate the relationship between Melampsora rust infection and drying rate, lightly rusted and heavily rusted shoots of P. trichocarpa were chosen using the basis described under "Inoculation Studies". In, addition, the effects of growth conditions on drying rates were investigated by the inclusion of shoots of wild pop-lars collected from the University Lands. The total length of each shoot was measured and divided into four quarters which were marked on the shoot. From the basal quarter (quarter U) and second quarter from the tip (quarter 2) 2 cuttings 30 cm. long were randomly selected. The experimental de-sign was, therefore, a factorial consisting of 5 poplars x 3 stumps x 2 quarters x 2 cuttings or 60 cuttings in toto. The cuttings were then excised from the shoot, placed in water and stored outside the laboratory until they had reached the saturation point. A l l exposed wood surfaces were coated with wax to ensure that loss of moisture took place only through the bark. The cuttings were set out on a laboratory bench supported at mid-point by small wooden stands so that air could circulate freely on a l l sides. As the buds broke, they were excised to prevent loss of water by transpiration and the scars were waxed over. A l l cuttings were weighed at five-day intervals and after each weighing were replaced in their previous .positions. Since previous experiments showed that over a few weeks losses in dry matter in cuttings did not exceed five per cent (see P. 28 ), a l l decreases in weight were attributed solely to =59-loss of moisture via the bark. At the end of the drying period, the oven dry weight from the cuttings was determined and the moisture content at each five-day interval calculated as a percentage of saturation. Dur-ing the course of the experiment the laboratory temperature averaged 74° F. and the relative humidity ranged from 48 to 54 per cent. Results are shown as mean moisture contents during the drying periods (Table 18) and as regressions of moisture content on time, i.e. drying rates (Table 19 and Plates 18 and 19) <> Inspection of the plottedcidata indicated equivalent variance and treatment as rectilinear regressions. A l l individual regressions were found to be highly significant. Highly significant differences occurred between the regressions of P. 'Robusta' and P. 'Regenerata', also between both of these and P. trichocarpa. There were no significant differences between heavily rusted, lightly rusted, u and wild P. trichocarpa. The regression effect was greatest in P. tricho-oarpa and least in P. "Regenerata0, i.e. moisture loss was slowest in the latter. It was noteworthy that P. trichocarpa differed much more from both hybrids than did the hybrids from each other, i.e«> the drying rates of the hybrids were somewhat similar compared to those of P. trioho-carpa. In a l l poplars except the wild P. trichocarpa. cuttings made from the lower part of the shoot had a significantly smaller regression effect than did those from the upper part of the shoot, i.e, the former lost moisture more slowly than the latter. The inconsistency in the wild P. trichocarpa was attributed to the disproportionately smaller diameter of the upper shoots compared to the lower shoots. There were no signifi-cant differences between the regressions of individual stumps within the poplars, i.e. there Was a relative uniformity within each type of poplar. If the drying rates in rooted plants were controlled by the same factors as in the cuttings in the laboratory, then under uniform conditions -60-TABLE 18 CHANGE IN MOISTURE CONTENT OF POPLAR CUTTINGS EXPOSED TO ROOM ATMOSPHERE Quarter 0 5 Drying period (days) 10 15 20 25 30 35 Moisture content (per cent saturation) P. trichocarua 2 100 84.9 59.6 47.8 37.3 4 100 89.9 74.2 59.9 48.5 38.3 Mean 100 87.4 66.9 53.9 42.9 P. trichocarre 2 100 90.1 64.6 47.9 40.7 (Rusted) 4 100 92.1 74.9 61.9 50.7 44.7 Mean 100 91.1 69.7 54.9 P. trichocarpa 2 100 89.6 51.4 (Wild) 4 100 94.1 78.7 65.6 48.4 34.5 27.5 Mean 100 91.8 65.O P. 'Regenerate1 2 100 91.0 69.8 54.8 45.1 34.4 26.7 4 100 94.2 83.6 74.3 64.6 54.0 42.7 38.0 Mean 100 92.6 76.7 64.5 54.8 44.0 • 34.7 P. 'Robusta' 2 100 91.1 68.0 51.4 40.9 30.2 23.9 4 100 92.7 78.7 65.7 54.1 42.8 34.5 30.3 Mean 100 91.9 73.4 58.5 47.5 36.5 29.2 Quarter 2 Quarter 4 = second quarter from tip. = basal quarter. -61-TABLE 19 REGRESSION OF MOISTURE CONTENT ON TIME IN POPLAR CUTTINGS EXPOSED TO ROOM ATMOSPHERE P. trichocarpa P. trichocarpa (Rusted) QUARTER 2 4 mean 2 4 mean REGRESSION FORMULA 99.28 - 17.09 X 100.29 - 12.74 X I Y Y = 100.80 - 15.80 X 101.73 - 17.01 X 100.51 - 12.00 X Y Y Y = 102.13 - 14.38 X P. trichocarpa (Wild) P. 'Regenerata' P. 'Robusta' 2 4 mean 2 A mean 2 f. mean 95.84 - 19.02 X 103.56 - 13.16 X Y Y Y = 96.64 - 11.98 X 98.81 - 12.86 X 101.83 - 9.39 X Y Y Y = 100.35 - H-10 X Y = 96.90 - 12.76 X Y = 96.52 - 9.39 X Y = 99.82 - 12.46 X Quarter 2 = second quarter from tip. Quarter 4 = basal quarter. -62-in the nursery the hybrids would lose moisture more slowly than P. tricho-carpa. Similarly the lower shoot would lose moisture more slowly than the upper. The, salient points in the infection process may be summarized as follows. (1) Above the critical moisture content, Cytospora canker growth varied as the moisture content. The hybrids had a lower critical bark moisture content for infection than P. trichocarpa. (2) In a l l poplars, bark moisture content was dependent on wood moisture content. (3) Given the same wood moisture content and the same conditions for drying, the hybrid cuttings lost water more slowly than did those of P. trichocarpa,. Therefore, as shown by the inocu-lation experiments, they took longer to become infected and i f uniform conditions of drying prevailed in the nursery, P. tricho-carpa shoots would become infected with Cytospora before the hybrids. The argument concerning the regions of the shoot is the same except i that the lower shoot appeared to have a higher c r i t i c a l bark moisture content than the upper. In view of the small difference in criti c a l bark moisture contents and the relatively large difference in regressions i t oould be reasonably inferred that the drying rate was the more important factor and that the upper shoot would become infected before the lower. While P. 'Regenerata' lost water more slowly, its incubation period (see Table 12) did not differ significantly from P. 'Robusta'. A possible explanation lies in the lower criti c a l bark moisture content of the latter which offset the faster drying rate, i.e. 53.2 versus 59.9 per cent. The lack of variation of stumps within poplars in drying rates was consistent with their lack of variation in the incubation periods. The lack of • . f -63-difference between the drying rates of the lightly rusted and heavily rusted P. trichocarpa indicated that any association which existed between Cytospora cankering and rust infection was not attributable to the drying rates of the shoots. Anatomical Characteristics of Poplar Shoots To further elucidate the moisture relations of the three poplars, attention was turned to the anatomical characteristics likely to have a functional significance in water storage, distribution, and retention within the shoot. Kennedy and Smith (1959) have reported that P. 'Regen-erata* had longer fibre tracheids than P. trichocarpa', and Meyer-Uhlenried (1958) found that the mean tangential diameter of the f i r s t early vessels of Tacamahaca poplars, including P. trichocarpa. was significantly differ-ent from that of the Aigeiros poplars including both P. 'Robusta' and P. •Regenerata'. These findings were suggestive of differences in moisture relations but there appeared to be few other reports in the literature on the comparative anatomy of the three poplars under investigation. Accordingly a critical investigation was made of their stem anatomy with the objective of relating anatomical characteristics to their moisture economies. The effects of the following factors were studied: type of poplar, region in the shoot (upper and lower), stump, and interaction of these factors. A balanced sampling design of the following construction was used: in each of the poplars a major shoot was randomly selected from each of three stumpsj each shoot was subdivided into quarters and two samples were randomly selected from the basal quarter (quarter 4 ) and from the second quarter from the tip (quarter 2). The design was, there-fore, a factorial similar to that used for the investigation of drying -64= . . . . . . rates. Study material consisted of nursery shoots cut after the end of the first growing season and in a dormant condition. Shoots were approx-imately 3 cm. in diameter at the base. Microtomed transverse sections were prepared from each sample and additional, longitudinal sections were made for supplementary observations. Differential staining was by fast green and safranin according to Johansen (1940) modified by doubling the safranin concentration. In each transverse section the following anatomical characteristics were sampled. Total diameter, bark thickness, pith diameter. Number of vessels and tangential lumen width. Number of tracheids and tangential lumen width. Number of xylem rays. Sieve tube area as a percentage of total inner bark area. Area occupied by the phloem fibres as a percentage of total inner bark area. Number of cells in the phellem and thickness of phellem. Number of cells in the phloem rays. Number of lenticels per unit bark area. Two subsamples of each of the above characteristics were taken in each of two randomly chosen points within the section in the following manners " Shoot diameters two diameters were measured at right angles. This and a l l other lineal measurements were made by stage or eyepiece micrometer. Pith: the pith was measured as the mean of two diameters at right angles. - 6 5 -Barks the bark was defined as a l l tissues exterior to the xylem and was measured as the sum of two bark thicknesses along a diameter. Two diameters were taken. Number of vessels % the number of vessels was counted in a superimposed field, approximately 0.19 sq. mm. in area. Four random samples were taken in each of the two positions. Width of vessel lumens to determine the reliability of tangential width as a measure of cross-sectional area, 20 vessels in each of the species, i.e. 60 i n toto were measured both tangentially and radially. No significant differences were found between species in the ratio tangential width/radial width. Thus tangential width was accepted as a proportional measure of vessel lumen area. Two samples of ten vessels were taken in each position, midway between the bark and the pith. Number of tracheidss the number of tracheids was counted in a superimposed field approximately .0027 sq. mm. in area. Four random samples were taken in each position. Width of tracheids: width of tracheid lumina was measured tangentially. Four samples of six tracheids were taken. Number of xylem rays: the number of xylem rays emerging at the perimeter of the xylem were counted in a standard length of circumference, approximately 1.2 mm. Sieve tube area: the areas of the bark occupied by the sieve tubes were measured and expressed as a percentage of the bark area interior to the periderm. -66-Phloem fibres, the area of the bark occupied by the phloem fibres was measured and expressed as a percentage of the total bark area interior to the periderm. Periderms the width of the phellem was measured in regions where i t was complete, I.e. where none had been eroded, the presence of residual epidermis be^ Lng taken as an indicator of this. Ridges in the bark were avoided. Two samples were taken in each position. The number of phellem cells was counted along two diameters. Phloem rayss the number of cells was counted from the f i r s t differentiated element to the last ray cell distinguishable. Number of lenticelss the number of lenticels was counted in two randomly chosen sections of the shoot 2 cm. long. Stem diameters were measured and the number of lenticels expressed on a unit bark area basis approximately 6.2 sq. cm. in area. The results of the analysis are summarized in Table 20 and are dealt with in detail below. Statistical analysis was carried out on a l l data. Differences cited in the following discussion were a l l signi-ficant at the five per cent level or higher. (a) Anatomical Characteristics of the Wood P. 'Robusta' and P. 'Regenerata' had wider piths than P. trichocarpa and since the total stem diameters of the poplars did not differ signifi-cantly the percentage of pith was greater in the hybrids. In a l l poplars the pith diameter was greater in the upper region of the shoot and, since the latter had a smaller diameter, the percentage of pith was greater than in the lower shoot. The stump variation was not significant. There was no difference between poplars in the number of vessels per -67-TABLE 20 ANATOMICAL CHARACTERISTICS OF BARK AND WOOD IN POPLARS SPECIES QUARTER CHARACTERISTIC Total diameter (mm) Pith diameter (mm) Bark width (%) Sieve tube area {%) No. ray cells Fibre area (%) No. phellem cells Width of periderm (u) No. of lenti-cels Vessel width (u) No. of vessels Tracheid width (u) No* of tracheids No. of rays P. trichocarpa 2 4 Mean 13.1 23.3 18.2 3.8 2.9 3.4 13.8 10.9 12.3 37.7 39.9 38.8 5.2 7.3 6.3 6.7 8.4 7.5 3.5 5.5 4.5 56.2 88.2 72.1 15.0 9.5 12.2 37.9 41.2 39.5 25.7 2^ .7 25.2 13.2 13.7 13.4 19.7 17.9 18.8 12.5 13.0 12.7 P. 'Regenerata' 2 4 Mean 14.2 22.3 18.2 5.2 4.7 4.9 13.2 11.7 12.4 49.3 51.8 50.5 7.5 11.8 9.6 13.9 14.1 14.0 5.3 8.3 6.8 112.7 152.2 132.3 14.7 11.0 11.8 49.6 53.8 51.6 21.7 18.9 20.3 15.8 15.0 15.3 14.3 14.8 14.6 12.3 11.4 11.8 P. 'Robusta' 2 4 Mean 14.5 22.3 17.5 5.6 4.6 5.1 13.0 11.4 12.2 48.4 54.7 51.5 8.4 9.6 9.0 15.0 15.8 15.4 5.3 8.1 6.7 103.6 142.2 122.8 9.1 7.1 8.1 51.6 54.0 52.7 22.7 18.9 20.8 14.4 14.3 14.3 13.3 13.8 13.6 11.7 10.7 11.2 -68= unit area of wood surface. However, vessels of P. trichocarpa had narrower lumina than those of the hybrids and thus the total vessel area was smaller in the former (Table 21)» The upper shoot in a l l poplars had more vessels than the lower, but these were smaller in diameter and thus there was no significant difference between total vessel area of the two regions of the shoot (Table 21). There was a significant interaction in vessel size between shoot regions and types of poplar due to the rela-tively small difference between regions in P. "Robusta'. Variation be-tween stumps within each poplar was significant in both vessel size and number. P. trichocarpa had more tracheids per unit area of wood surface than the hybridss however, lumen widths were not significantly different. This, together with the data for vessels, implied that in the xylem of P. trichocarpa total tracheid area was greater but total vessel area was smaller than in the hybrids. Stump variation was significant in both number and diameter of tracheids (Plates 20 and 21). (b) Anatomical Characteristics of the Bark There were no significant differences between the poplars in bark width expressed as a percentage of total shoot diameter; on the other hand, the bark percentage of the upper shoot was greater than that of the lower. Stump variation was not significant. The percentage of the bark area occupied by sieve tubes was greater in P. 'Robusta' and P. 'Regenerata' than in P. trichocarpa and was greater in the lower shoot of a l l poplars than in the upper. Stump variation was not significant. There was no difference in the number of rays between poplars, between shoot regions, nor between stumps. However, phloem rays in the hybrids extended farther towards the cortex and were broader tangentially than in -69-TABLE 21 TOTAL VESSEL AREA IN GROSS-SECTIONS OF POPLAR SHOOTS Quarter Total vessel cross-sectional area (u ) P. trichocarpa 2 4 Mean 369.1 419.3 393.2 P. 'Regenerata' 2 4 Mean 533 .8 547.1 540.5 P. 'Robusta' 2 4 Mean 604.4 551.1 577.7 -70-P. trichocarpa. The percentage of the bark area occupied by fibres was greater in the hybrids, but there was no difference between the shoot regions. Stump variation on the other hand was significant. There were marked differences between the poplars in fibre arrangement. P. 'Robusta1 had three distinct rows of fibres - an Inner continuous sheath within the secondary phloem, a middle one roughly bounding i t and a zone of outer bundles bounding the primary phloem. In P. 'Regenerata8 there were two well developed fibre zones, but in P. trichocarpa there were usually only two poorly developed zones. P. trichocarpa had fewer layers of phellem cells and a thinner periderm than either of the hybrids. While the number of layers of cells was the same in both hybrids, the actual thickness of the periderm was greater in P. 'Regenerata' than in P. 'Robusta'. Thus, in order of periderm thickness P. 'Regenerata' was greatest, P. 'Robusta' next, and P. trichocarpa least. It is important to note that the periderm of the latter was only one-half as thick as that of P. 'Regenerata'. P. 'Robusta' had fewer lenticels per unit bark area than did either P. 'Regenerata' or P. trichocarpa. The latter poplars were not significantly different from each other. The frequency of lenticels in P. trichocarpa was 50 per cent greater than In P. 'Robusta'. Similarly, the upper region of the shoot in a l l poplars had more lenticels per unit bark area than the lower. There was also a small interaction of regions and forms of poplar due to the disproportionately greater frequency of lenticels in the upper shoot of P. trichocarpa. There was, however, no significant variation due to stumps within the poplars (Plates 22, 23, and 24). To summarize the data for both wood and bark the hybrids had larger pith, wider vessel lumina, a larger sieve tube zone, longer and wider =71-phloem rays, larger bark fibre zone, thicker periderm and fewer lenticels than P. trichocarpa. The significance of these features from the view-point of their effect on moisture relations may be considered in the light of their functional properties. The pith consisted of loosely arranged, thin-walled parenchyma cells suggesting a high capacity for water storage. This was shown by measur-ing moisture contents of shoot sections containing different percentages of pith and which had been saturated with water. Those with a greater pith percentage had a greater moisture content on a dry weight basis (Table 22). The wood as a whole is concerned in the vertical, radial and tangen-t i a l movement of water in the stem. According to Curtis and Clark (1950) water is carried largely through the vessels of angiosperms rather than through the fibre tracheids. If this is the case, the size of vessels . should be an important factor in water movement and storage. Considering the forces of friction and the immobilization of water by binding forces in the cel l walls, i t would appear that wider vessels would conduct water more rapidly and that they would not only contain more water but a smaller percentage of i t would be bound in the walls than in narrow vessels, i.e. a larger percentage of water would be free to move. If, on the other hand, i t is assumed that tracheids conduct a significant amount of water then the same considerations should apply, i.e. wider tracheids would contain more mobile water and would conduct i t more rapidly. In addition, since tracheids are individual cells the longer tracheids would conduct water in a vertical direction more rapidly than the shorter ones since fewercend walls would intervene in a given distance. In the bark of the poplars being studied, i.e. six-month old shoots, the cortical cells, primary phloem and secondary phloem appeared to be -72= TABLE 22 WOOD MOISTURE CONTENTS OF UPPER AND LOWER STEM IN POPLAR SHOOTS Quarter Pith width (% of shoot) Per cent moisture (O.D.W.) of saturated shoot trichocarpa 2 4 2 9 . 0 12.4 396.6. 231.6 'Regenerata1 2 4 3 6 . 6 2 1 . 1 4 9 6 . 6 3 1 3 . 6 'Robusta' 2 4 3 8 . 6 2 9 . 1 5 5 9 . 0 2 6 4 . 6 -73-fully functional. Since the cortex parenchyma .was. adjacent to the sieve tubes i t was possible for water to diffuse between them. Thus If the sieve tubes were fluxed with water, as indicated by the mass flow theory at least, they could serve as irrigation channels for the cortex cells. Thus the greater the number of sieve tubes in relation to the cortex parenchyma the more effectively could turgidity be maintained in the latter. The bark fibres might also act to some extent as reservoirs of water in the bark. Although the lumina were narrow, a certain amount of diffusible water may have been present in their walls. If the vascular rays are the most likely routes for radial water movement between xylem and cambium then the more numerous or longer and larger the rays,, the greater the capacity for water movement. The function of protection and prevention of moisture loss from the inner bark tissues has been attributed to the periderm. Thus greater thickness of the periderm and/or greater number of phellem cell layers in the periderm should result in smaller moisture loss by evaporation from the bark. Since lenticels are generally held to permit gaseous exchange through the periderm, the greater the frequency of these in the bark the more water vapour could move through them from the cortex to the atmosphere when a vapour pressure gradient exists.. Comparing the anatomical findings with the results of the drying rates study (see Page 59) i t is seen that P. 'Regenerata* had the thickest peri-derm and the slowest drying rate. P. 'Robusta' had fewest lenticels and the next thickest periderm and its drying rate was slower than that of P. trichocarpa.* The latter, which had the most lenticels and the thinnest periderm, also had the fastest drying rate. Similarly, cuttings from the upper part of the shoot had thinner periderms and faster drying rates than those from the lower. Thus, the differences in drying rate between the -74-poplars were at least partially explicable on anatomical grounds. A comparison of the anatomical results with the a r t i f i c i a l inoculation studies showed that the hybrids had a greater sieve tube zone and a greater fibre zone than P. trichocarpa while their crit i c a l bark moisture contents for infection were lower. If, as previously postulated, the sieve tubes and fibre lumina provided water storage and replaced water losses in the cortical parenchyma cells, then the greater the proportion of such storage cells the drier the bark as a whole would be before the turgidity of the parenchyma cells was reduced and their resistance to infection decreased. This may be likened to a buffer effect of water-storing elements protect-ing the living cortex cells from desiccation. The anatomy of the bark, therefore, suggested a basis for the lower cr i t i c a l moisture contents in the hybrids, and by inference, their greater resistance to Cytospora infection. If the anatomical results are examined in the light of the incubation periods for the different poplars (see Page 39), i t is seen that the hybrids which took longer to become infected had larger vessels and larger pith. If, as previously suggested, these characteristics gave the hybrids greater water storage in the shoot then i t would take longer for their bark to dry to the same moisture content as that of P. trichocarpa. This conclusion seemed warranted in view of the highly significant regressions which were found of bark moisture content on wood moisture content. It should be noted that the quantity of water storage was a separate factor in the incubation period from the rate of moisture loss as influenced by periderm thickness and number of lenticels. Thus the longer incubation period of the hybrids may have been attributable to both a greater volume of stored water and to slower rate at which i t was lost. -75-Cuttings made from the lower part of the shoot arrested the canker more rapidly than those from the upper part when they were plaoed in water. Also the lower shoot had larger vessels and a larger sieve tube zone. These characteristics suggested a reason for more rapid rehydration of the tissues in the lower shoots. Thus turgidity of their bark cells could have been restored more rapidly in the lower than ihi the upper shoots.-Apart from moisture relationships, stem anatomy in the three poplars may be correlated with Cytospora canker in other ways. The hybrids which in the field showed much greater resistance than P. trichocarpa also had thicker periderms. Since the fungus appeared to be a wound parasite, a thicker periderm should have reduced the chances for hyphal penetration. Also, as the a r t i f i c i a l inoculation studies showed, the canker grew more slowly in the hybrids, even when an entry for the fungus was provided. The hybrids had a greater proportion of fibres in the bark than had P. trichocarpa so that this may have retarded the growth of the fungus through the tissues. This surmise was supported by the appearance of bark cankered by Cytospora in which strands of fibres seemed untouched while other tissues were decomposed. It was difficult to assess the importance of these mechanical factors in resistance compared with those due to moisture content. It was probable that they were complementary. As shown by the inoculation experiments, when the moisture content was above the criti c a l level no infection took place even when the periderm was removed. However, even when the moisture content was low, the thicker periderm of the hybrids in the nursery may have lessened the chances of fungal entry. In addition, the more numerous fibres in the hybrids may have significantly retarded growth of the fungus so that when a higher moisture level was restored the canker was confined =76= to a very small area of the bark.' On the other hand, the evidence should not be overlooked that the moisture content of the hybrid was less likely to have fallen to the criti c a l level. Shoot Moisture Content of Poplars in the Field In order to relate field conditions to the findings of the foregoing laboratory investigations, a moisture sampling program was carried out in which samples were taken at approximate intervals of'six to eight weeks beginning in July, 1958, and continuing until December, 1959• (Bark samples were collected beginning October, 1958.) Three major shoots from three stumps of each of the poplars were selected at random at each sampling. , These were marked into quarters and two moisture samples taken from quarter 2 and quarter 4« Each sample comprised a disc approximately 1 cm. thick. The bark was peeled from this and the moisture content of bark and wood determined separately. Results were expressed on a saturation basis. Standardized climatic conditions were chosen for each sampling to minimize possible variation due to atmospheric humidity and diurnal rhythm in moisture content. In summer, samples were taken in the early morning before sunrise and the leaves immediately removed. In winter, samples were taken in the early morning when either rain was falling or the at-mospheric humidity was almost 100 per cent. Results are shown in Table 23. Comparison of the mean bark moisture values for a l l poplars showed that significant differences occurred between a l l months except December and February. The values of July, September, April, October and December were in descending order of magnitude. Compar-ing the three poplars, P. trichocarpa had the highest mean - 84..0 per cent, P. "'Regenerata' was next with 81.7 per cent, and P. 'Robusta' had the low-est with 78.3 per cent. The differences between these, although signi-ficant, were less than six per cent. A comparison by individual sampling -77-TABLE 23 MARCH OF MOISTURE CONTENT (%) SATURATION) IN POPLAR SHOOTS Wood Moisture Content 1958 1959 July 1 Sept. Oct. Dec. Feb. May July Sept. Oct. Dec. P. trichocarpa 54.0 72.5 55o6 63.3 63.8 55.6 85.2 77.2 34.2 62.9 P. 'Regenerata' 57.0 63.0 34.8 69.4 75.8 83.0 93.4 63.7 35.5 Y7.9 P. 'Robusta5 64.O 5 6 . 7 37.5 72.9 75-0 76.8 97.8 58.9 37.5 74.6 LSD Poplars (P < . 0 5 ) 6.2 Upper shoot 63.3 73.7 4 1 . 9 63.0 6 5 . 7 6 5 . 9 96.8 68,9 35.5 6 4 . 2 Lower shoot 53.3 54.2 43.4 74.6 77.4 76.3 81.9 64.3 36,9 79.3 LSD Quarters ( P < . 0 5 ) 5.3 Average 58.3 63.9 4 2 . 6 68.8 71.5 71.1 89.3 66.6 36.2 71.7 Bark Moisture Content P. trichocarpa - 86.1 71.5 77.6 88.8 97.7 94.3 74.1 82.3 P o 'Regenerata' 73.4 70.7 73.4 87.3 98.5 94.8 78.2 77.3 P o 'Robusta' 6 8 . 4 71.4 69.0 86.0 97.8 89.4 68.3 75.8 LSD Poplars ( P< o 0 5 ) 3.1 Upper shoot 78.8 72.0 73.8 89.5 99.9 95.2 74.6 75.8 Lower shoot 73.7 70,5 72.9 85.3 96.3 90,5 73.7 81.0 LSD Quarters ( P < . 0 5 ) 2.6 Average 75.9 71.2 73.4 87.4 98.1 92.9 73.5 78.8 ^Samples taken in afternoon Instead of early morning. -78-times showed that P. trichocarpa had a significantly higher value than one or both hybrids on five occasions, but only in October did the difference equal or exceed 10 per cent. In the remaining three sampling times there was no significant difference between the poplars. Comparison of the wood moisture content of the individual sampling dates showed that the value for October of both years was significantly lower, and July, 1959 higher than the other dates which did not differ among themselves. Here the mean value for each poplar was of l i t t l e interest since the statistical tests showed a highly significant interaction between sampling dates and poplars. This interaction arose principally from the fact that while P. trichocarpa was significantly lower than hybrids in December, February, April, and July, i t was equal to or higher than the hybrids in September and October. Actual differences between P. trichocarpa and the hybrids exceeded 10 per cent. Comparisons of the two regions of the shoots, i.e, upper (quarter 2) and lower (quarter 4-) showed that the former had a higher mean bark moisture content, i.e. 82.4. versus 80.5 per cent. The difference, though significant, was relatively small. In the case of wood moisture content, a highly signi-ficant interaction occurred between dates and shoot regions similar to that encountered in differences between poplars. Moreover, the origin was the same in that the December, February and April values of the upper shoot were lower than those for the lower shoot, but in July, September, and October they were equal to or higher than those of the lower shoot. Differ-ences approached or exceeded 10 per cent. Values for both regions reached their minima in October. The variation between stumps within species was not significant. On the basis of an eighteen-month sampling period the evidence was that in a l l the poplars the wood moisture content minimum occurred in -79= Octobero It was well defined, being at least 20 per cent lower than the values for the preceding and succeeding sampling dated That this may be the general rule was indicated by the corroborating data of Gibbs (1957) which showed that wood moisture minima occurred in late September in Populus tremuloides and P. deltoides trees growing in eastern Canada. These minima were also well defined by a sharp drop preceding and a sharp rise succeeding them. Furthermore, other hardwoods sampled by Gibbs showed minima during September to October, e.g. species of Acer, Salix„ Betulao Juglans and UlmuSo If this was the rule for poplars then i t was reasonable to conclude that shoot moisture content was more likely to be reduced to the criti c a l level for Cytospora infection in late autumn than at other times of the yearD In the three-year observation period at the nursery the only major outbreak did, in fact, occur in early winter (Nov-ember to Decembers 1956)o The question then arose? since the late autumn wood moisture content of the hybrids f e l l to the same level or lower than that of P. trichocarpa how could this fact be reconciled with their observed greater resistance in the nursery? Part of the answer may have lain in the fact that the critic a l bark moisture contents of the hybrids were lower, as indicated by the inoculation experiments. Thus while the wood became as dry or drier than that of P. trichocarpa the bark moisture did not f a l l to the critical level. Secondly, examination of the data showed that following the minimum, the hybrid wood moisture regained a higher level than that of P. trichocarpa and maintained its superiority throughout the following year. This implied a more rapid refilling of the hybrid wood with water and i t may, therefore, be argued that even i f the wood moisture content of a l l poplars had fallen to the criti c a l level in late autumn, the hybrids could have more rapidly regained and maintained a moisture level above the -80-minimum for Infection., Since the a r t i f i c i a l inoculation experiments showed that canker growth was halted when cuttings were placed in water, i t may be reasoned that cankers present in field shoots would also be halted when sufficient moisture was supplied by the roots 0 Thus, assuming that infection did take place In the hybrids during late autumn, such fungus growth as occurred would have been halted when the wood mois-ture content rose above the critical level in the ensuing month. In P. trichocarpa on the other hand, the rise may have been insufficient to bring the level above the threshold for infection. Thirdly, infection by Cytospora in the field was presumably unlikely to be instantaneous as soon as the bark lost sufficient moisture. Moreover, i t may have taken longer for hyphae to penetrate the hybrids due to their thicker periderm and also canker growth may have been slower, as indicated by the inocula-tion experiments, due to their more fibrous bark. Since the hybrids would tend to regain a level above the threshold more rapidly than P. trichocarpa their chances for Infection and extensive cankering would have been smaller than those of P. trichocarpa. Since the autumnal minima for wood moisture contents were the same for both regions of the shoot, and the lower shoot refilled more rapidly than the upper, the above arguments wouid also predict a greater cankering in the latter. Thus i t would appear that the least resistant host material was the upper shoot of P. trichooarpa and the most resistant was the lower shoot of the hybrids. The occurrence of cankers fi r s t in the upper shoot of P. trichocarpa was in fact observed during the nursery outbreak. To theorize on the cause of moisture fluctuations in the poplars, i t was necessary to assume that these were attributable either to variations in moisture uptake, or in moisture loss or in both. The sharp autumnal drop could be more plausibly attributed to a decrease in water uptake, i f -81-for no other reason than the transpiration conditions were less favourable than in the preceding month and in themselves would offer no evidence for a sudden Increase In moisture loss. If, on the other hand, the rate of water absorption by the roots decreased in late autumn below that of trans-piration, the water In the vessels would be reducedo After defoliation, moisture loss should be limited to evaporation from the baric This loss has been found by Weaver (1919) to be relatively small and i f root absorption exceeded i t , a refilling of the vessels would have occurred. This was the case judging from the rapid rise in wood moisture content. The differences in the wood moisture content of poplars in the growing season may have been caused, therefore, by differences in the absorption rate and/or differences in the transpiration rate and in the leafless season by differences in absorption rate and/or differences in losses via the bark. The anatomical studies and drying experiments have provided evidence that bark moisture losses are one factor in the water balance and i t is possible that differ-ences in water absorption were another. Comparison of the upper and lower shoot showed that there was more moisture in the lower shoot during dormancy but the same or less during growth. If, following defoliation, root pressure became the main force lifting water up the shoot but was insufficient to force water to the top of the shoot, the latter would be less hydrated than the lower. The consequence of this would be the desiccation of the bark as suggested in the previous discussions of bark/wood moisture relations (see Page 57). The difference in moisture content between shoot regions would be further enhanced by the greater loss by evaporation in the upper shoot. During growth, by contrast water is drawn to the top of the shoot by transpiration from the upper crown and the water stream would be then distributed equally along the axis. If, indeed, there was tension in the vessels, more of <=82.«? these could be devoid of water in the lower shoot* The conclusion was, therefore, that the greater drying rate and smaller hydration of the upper shoot would lead to a smaller water content during dormancy but that during growth the transpiration stream was sufficient to offset the losso On the other hand, more vessels in the lower shoot could contain water during dormancy owing to the greater hydration and smaller loss but during transpiration a greater number of vessels could be empty. The data for bark moisture content during the sampling period of sixteen months showed a yearly maximum occurring in July and a decline to a minimum in December and February. The trend was similar to that found by Gibbs in P. deltoides growing in eastern Canada except that the maximum for the latter occurred somewhat earlier, ioe. June. Similar trends occurred in Salixo Betula. Juelans and Fraxinus. Comparison of wood moisture and bark moisture trends in the three poplars at the nursery showed that the maxima for both values occurred in July and that both showed a marked drop in October-as would be expected from the correlations of bark and wood moisture content. If lowering of the bark moisture content was the immediate cause of Cytospora infection, then late autumn and early winter were evidently the most likely periods for this to occur. However, this would also depend on the factors promoting desiccation, e.g. air temperature, solar radiation, relative humidity and wind. Thus for a given wood moisture content, more powerful drying condi-tions would produce a drier bark than less powerful ones. The balance between wood moisture content and bark moisture content loss was, therefore, the controlling factor in Cytospora infection. Evidence so far is that , this was more likely to occur in P. trichoearpa than the hybrids and in late autumn than at other times. However, at any time and in any of the poplars when conditions arose which would reduce wood moisture and desiccate -83-the bark sufficiently, i t could be predicted that infection would take placeo It was noted in the discussion of the cri t i c a l moisture contents for infection that Butln found crit i c a l values for P. deltoides cuttings collected in October ranging from 78 per cent to 82 per cent of the existing cutting moisture content in contrast to the values found i n the nursery poplars ranging from 24. per cent to 4-0 per cent of total saturation. If, as indicated, shoot moisture content minima for poplars in general occur in early winter and i t is assumed that the values f a l l within roughly the same range, then the nursery values would be somewhat higher than those found by Butin, the latter being approximately 80 per cent x (24 per cent to 40 per cent) = 19 to 32 per cent of saturation., SUMMARY This report has presented evidence to show that the differences in resistance to Cytospora canker which were observed in three forms of poplar growing at a nursery were correlated with their moisture contents, and that under the same conditions the poplars showing the higher resistance, i>e. the hybrids, had the higher moisture content than the less resistant P. trichocarpa. External factors which reduced moisture content, e.g. high temperature, low relative humidity, low soil moisture, also increased Cytospora canker growth. Internal factors which favoured the maintenance of a higher moisture content, e.g. slower moisture loss from the shoot and more rapid refilling of the shoot, were more pronounced in the hybrids than in P. trichocarpa, The internal factors were in turn correlated with anatomical characteristics which, in the hybrids, suggested a smaller water loss from the bark, a more rapid translocation, and a greater storage of water in the wood. In addition, the hybrids had lower cr i t i c a l moisture levels for -84-infection than P. trichocarpa. Since their drying rates were slower than P. trichocarpa„ their incubation period was longer. With one exception the explanations for differences in resistance between the poplars applied to differences between the /upper and lower shoot. The upper shoot was found to be less resistant to Cytospora in the nursery, to have a lower moisture content during the dormant period, to dry more quickly, and to possess fewer anatomical mechanisms for water retention and translocation than the lower shoot. The exception to the parallelism was that the critical moisture content for infection of the upper shoot was lower. However j, the differences were small relative to the much faster drying rate of the upper shoot. Shoot moisture contents were significantly lower in early winter than at other times of the year, which might explain the occurrence of the nursery outbreak in November and also the findings of other investi-gators regarding different canker organisms on poplar. Furthermore, the most susceptible poplar, P. trichocarpa. had a lower shoot moisture content throughout the dormant season than either of the hybrids, as had the more susceptible upper region of the shoot. The increase of Cytospora growth in proportion to temperature, within limits, was compatible with the fact that higher temperatures also increased cankering of the shoot. Furthermore, in view of the fact that the disease outbreak occurred during early winter i t was significant that some growth of Cytospora took place at temperatures as low as 0° C. A synthesis of the evidence from the investigation suggested that a general etiological picture could be described as follows. The hosts A poplar whose anatomical characteristics suggested a poor moisture retention, storage and translocation and whose rate of moisture absorption was low. The environments Low available soil moisture, temperature 25° G0 approximately, low relative humidity,, some wind. The seasons Early winter extending through early spring. As a generalization, the evaluation of host characteristics for determining tree resistance to facultative disease organisms would seem worth stressing. A better criterion for programs of tree breeding may be provided by such characteristics than by others now in use. The identi-fication of the operative factors should enable the forest geneticist to breed for these characteristics from selected parents, and to obtain with reasonable certainty the desirable resistant progeny. By contrast, breed-ing from parents whose only selection characteristic is freedom from the disease, at best only achieves the same results over a long test planting period and at worst may result in the finding that the resistant property is not genetically controlled. BIBLIOGRAPHY 1. Addicott, F. T. 1944.. Some anatomical effects of moisture stress in nursery seedlings of guayule. Bot. Gaz. 106s 208=214.. 2. Ashton, Floyd M. 1956. Effect of a series of alternating high and low soil moisture contents on the rate of apparent photo-synthesis in sugar cane. Plant Phys. 31s 266-274. 3. Auchter, E. C. 1923. Is there normally a cross-transfer of foods, water and mineral nutrients in woody plants? Maryland Agric. Expt. Sta. Bull. No. 257s 33=62. 4. Barnett, H, I. 1955. Illustrated genera of imperfect fungi. Burgess Publishing Co., Minneapolis. 5. Bier, J. E. 1939. Septorla canker of Introduced and native hybrid poplars. Can. Jour. Res. (Sec-. C) 17(6)s 195-204. 6. Bier, J. E. 1959. The relation of bark moisture to the development of canker diseases caused by native facultative parasites. 1. Cryptodiaporthe on willow. Can. Jour. Bot. 37s 229=238. -86. 7. Bier, J. E. 1959 A. The relation of bark moisture to the development of canker diseases caused by native facultative parasites. • 2» Fusarium on black cottonwood. Can. Jour. Bot. 37; 781-788, 8. Bier, J. E„ 1959 B. The relation of bark moisture to the development of canker diseases caused by native facultative parasites. 3<> Cephalosporins; on western 'hemlock. Can. Jour. Bot. 37s 1140= 1142. 9 c Brown, H. P., A, J, Panshin and C. C. Forsaith. 1952, Textbook of Wood Technology, Vol. 2. - McGraw-Hill Book Co., Toronto. 10. Butin, H. 1955o Uber'den Einfluss des Waasergehaltes der Pappel auf ihre resistenz gegenuber Cytospora chrysosperma (Pers.) Fr. Phytopath. Zeitsehr. 24s. 245-264. 11. Butin, H. 1956» Untersuchungen tiber Resistenz und Krankheitanfalligkeit der Pappel gegenuber Dothlehiza populea Sacc. et Briard. Phytopath. Zeitschr. 28(4)« 353=374, 12. Christensen, C. M. 1940. Studies on the biology of Valsa sordida and Cytospora chrysosperma. Phytopath. 30s 459=475, 13. Crafts, A, S.„ H, B, Currier and C.'R. Stocking. 1949. Water in physiology of plants. Chronica Botanica Co., Waltham, Mass. 14. Curtis, 0. F., and D. G. Clark, 1950, Introduction to plant physio-logy. McGraw-Hill Book Co., Toronto. 15. Gaumann, Ernst. 1950, Principles of plant Infection. Hafner Publishing Co., New York. 16. Gibbs, R. Darnley. 1935. Studies of wood. III. On the physiology of trees with special reference to the ascent of sap and move-ment of water before and after death. Can, Jour. Res. 12s 761-787. 17. Gibbs, R. Darnley. 1942. Studies in tree physiology. III. The effect of the method of cutting on the water content of twigs. A note on a paper by MacDermott. Can. Jour. Res. (Sec. C) 20s 236-240. 18. Gibbs, R. Darnley. 1957, Patterns in the seasonal water content of trees. Symposium on the physiology of forest trees, Harvard Forest. The Ronald Press Co., New York. 19. Harper, V. L. 1936. Effects of fire on gum yield of long leaf and slash pines. U.S.D.A. Tech. Bull. No. 510. 20. Helmerick, R. H. and Pfeifer, R. P. 1954. Differential varietal response of winter wheat germination and early growth to controlled limited water conditions, Agron. Jour. 46s 560= 562. 21. Hubert, E. E. 1920. Observations on Cytospora chrysosperma in the northwest. Phytopath. 10s 442-447. -87-22. Johansen, D. A. 1940. Plant microtechnique. 1st Ed. MeGraw H i l l Book Co., Toronto. 23. Kennedy, R. W. and J. H. G. Smith. 1959. The effects of some genetic and environmental factors on good quality in poplars. Pulp and Paper Magazine of Canada. February 1959. T35-36. 24. Long, W. H. 1918. An undeseribed canker of poplar and willows caused by Cytospora chrysosperma. Jour. Agr. Res. 13: 331-345. 25. MacDermott, J. J. 1941. The effect of cutting on the moisture content of samples. Am. Jour. Bot. 28s 506-508. 26. MacDougal, D. T. 1938. Tree growth. Chronica Botanica Co., Waltham, Mass. 27. Meyer, B. S. and D. B. Anderson. 1952. Plant physiology. 2nd Ed. D. von Nostrand Co. Inc., Princeton, K.J. 28. Meyer-uhlenried, K.H. 1958. Untersuchungeh uber die Vererbung einer anatomischen Merkmals bei Kreuzungen von Pappeln verschied-ener Sectionem Der Zuchter 28(5): 209-216. 29. Mooi, J. S. 1948. Kanker en taklhsterving van der wilg veroonzaakt door Nectria gallieena en Cryptodiaporthe salicina. Thesis, Gitgeverij and Drukkerij, Baarn, Hollandla. 30. Moss, E. H. 1922. Observations on two poplar cankers in Ontario. Phytopath. 12: 425-427. 31. Muller-Stoll, W. R. and Ursula Hartman. 195°. Uber den Cvtospora Krebs den Pappel (Valsa sordida Nitshke) uhd die Bedigungen fur eine parasitare Ausbreitung. Phytopath. Zeltschr. 16s 443-478. 32. Munch, E. 1909. Untersuchungen uber Immunitat und Krankheit-sanfalligkeit der Holzpflanzen. Katurw. Zeltschr. Forst-u Landwirtsch. 7s 54-75, 87-114, 129-160. 33. Priestly, J. H. and L. M. Woffenden. 1922. Physiological studies of plant anatomy. V. Causal factors in cork formation. New Phytol. 21s 253-268. 34. Schmidle, A. 1953. Die Cytospora - Krankheit der Pappel und die Bedingungen fur ihr Auftreten. Phytopath. Zeltschr. 21(1): 83-96. 35. Schmidle, A. 1953 A. Zur Kenntnis der Biologie und der Pathogentitat von Dothichiza populea Sacc. et Briard den Erregen eines Rindenbrandes der Pappel. Phytopath. Zeltschr. 21(2)s 190-209. 36. Schreiner, E. J. 1931. Two species of Valsa causing disease in Populus. Am. Jour. Bot. 18s 1-30. -88-37. United Nations, Food and Agriculture Organization. 1955« Report -of the Standing Committee on the application to the genus Populus of the rules laid down In the International Code of Nomenclature for cultivated plants. Proc. U.N., F.A.O. International Poplar Commission, 8th Session, Madrid. 38. Upchurch, R. P., M. L. Peterson and R. M. Hagan. 1955. Effect of soil moisture on the rate of photosynthesis and respiration in Ladino clover. Plant Phys. 30s 297-303. 39. Van Meiden, H. A. and H. Van Vloten. 1958. Roost en Schorsbrand als Bedreiging van de Teelt van Populler. Nederlands Bosbouw Tijasehrift 30s 9. 40o Vloten, H. Van. 1952. Evidence of host-parasite relations by experiments with Phomopsis pseudotsugae Wilson. Scot. For. 6(2)s 38-46. 41. Waterman, A. M. 1957. Canker and die-back of poplars caused by Dothlchlza ponulea. For. Sci. 3s 175-183. 42. Wadleigh, C. H. and H, D. Gauch. 1948. Rate of leaf elongation as affected Dy the intensity of the total soil moisture stress. Plant Phys. 23s 485-495. 43. Weatherby, P. E., A, J. Peel and G. P. H i l l . 1959. The Physiology of the sieve tube. Preliminary experiments using aphid mouth parts. Jour. Exp. Botany 10(28)s 1-16. 44. Weaver, J. E. and A. Morgensen. 1919. Relative transpiration of coniferous and broadleaved trees in summer and winter. Bot. Gaz. 68s 393-424. 45. Weimer, J. L. and L. L. Harter. 1921. Wound cork formation in the sweet potato. Jour. Agr. Res. 21s .637-647. 46. Wright, E 0 1957. Cytospora cankers of cottonwood. Plant Dis. Rep. 41*. 892-893. , Plate 1 Natural Cytospora cankers on Populus trichocarpa cuttings occurring under storage conditions. Note infection courts at nodes, lenticels and small bark injuries. Plate 2 Appearance of poplar nursery in the summer following sever winter infection of Cytospora chrvsosnerma. Open spot has been produced by individuals which failed to survive. Remainder of trees are healthy. Plate 3 Relationship of root formation and Cytospora canker in 4-month old Populus trichocarpa cuttings from a plantation. Cutting on right has best root development and no canker; cuttings second and third from right have fewer roots, and are cankered (indicated by white marks). Natural seedling on left is for comparison of root systems, and is not cankered. Plate 4 inoculations of Ponulus spp. with Cvtospora. Upper lation is a check wound in each case. Plate 5 Figures 1-3 Cytospora canker on Populus trichocarpa growing in greenhouse. In each case the canker was arrested and wound periderm formed. Plate 6 Method of taking moisture samples from one-year old Populus  trichocarpa shoots. Double-bladed hacksaw permitted two simultaneous cuts to be made at each stroke. Wooden vise held stem firmly but without damage to bark. Plate 7 Fruiting structures of Cytospora chrysosperma. Figure 1. Spore horns emerging from bark of Populu3 'Regenerata'. (X 6) Figure 2 Pycnidia on cuttings of P. trichocarpa which had been Inoculated with Cytospora'. Plate 8 Cytospora chrysosperma hyphae in bark of Populus trichocarpa. Figure 1 Intracellular within cortex. Figure 2 Within sieve tube. Figure 3 Intercellular between cortex and secondary phloem. (X 126) Plate 9 A r t i f i c i a l inoculations of Populus trichocarpa with Cytospora. Figure 1 Canker has advanced from original inoculation wound (outlined In black) and girdled a young shoot. Figure 2 Canker (outlined in white) has extended beyond wound only in dry cuttings. No canker growth occurred in cuttings placed in water. 1 0 0 r T I M E ( D A Y S ) J Plate 10 Growth of Cytospora canker in inoculated cuttings of Populus trichocarpa subjected to different watering regimes. Numbers refer to cuttings. \ 0 10 2 0 5 0 7 5 1 0 0 TEMPERATURE C O INITIAL MOISTURE CONTENT ( %>) Plate 11 Growth of Cytospora canker In inoculated poplar cuttings given different moisture and temperature treatments. -5 0 5 10 15 2 0 2 5 3 0 35 TEMPERATURE • C _ Plate 12 Growth rate of Cytospora oh £ per cent P.D.A. in the dark at different temperatures. R . H . 1 0 0 % Plate 13 Cytospora canker growth in poplar cuttings given different moisture and relative humidity treatments. Plate L4 Arrest of cankers in Populus trichocarpa cuttings inoculated with Cytospora. Figure 1 Method of inducing adventitious roots in cankered cuttings. Figure 2 Detail of roots produced by cankered cuttings. Maximum root length is 30 mm. Figure 3 Healthy cuttings, two months following potting. Arrested cankers are visible on lower main shoot. Figure U Root systems of cuttings which grew vigorously following potting. Plate 15 Arti f i c i a l drought experiments with Populus trichocarpa• Figure 1 Method of Inoculation. Figure 2 Root systems of droughted plants (left) and undroughted plants (right). Roots were balled up due to their growth In pots. Figures 3 & 4 Wound periderm produced by undroughted plant (left) and droughted plant (right). Note heavy ridge in former, healing out the inoculation and weak periderm of droughted plant permitting latent canker to establish. Plate 16 Art i f i c i a l drought experiments with Populus hybrids. Figure 1 Inoculated plants which have been droughted but not exposed to low relative humidity. They have not been killed, although some defoliation has occurred. Figure 2 Inoculated plants exposed to both drought and low humidity. They have been killed by Cytospora. Figure 3 Appearance of droughted plants after a period of dormancy. 20 3 0 40 50 60 70 80 90 100 WOOD MOISTURE CONTENT Plate 17 Regressions of bark moisture content on wood moisture content in six-month old Populus cuttings collected in f u l l growth. Plate 18 Regression of moisture content on time in Populus cuttings exposed to room atmosphere. Vertical scale, percentage of saturation; horizontal scale, days of exposure. Q2 = cuttings from 2nd quarter, 04 = cuttings from 4-th (basal) quarter. Figure 1 P. 'Regenerata1. Figure 2 P. 'Robusta'. Figure 3 P. trichocarpa. lightly rusted. Figure 3. P. trichocarpa (nursery) Plate 19 Regression of moisture content on time in Populus cuttings exposed to room atmosphere. Vertical scale, percentage of saturation; horizontal scale, days of exposure. Q2 = cuttings from 2nd quarter, Q4 = cuttings from 4th (basal) quarter. Figure 1 P. trichocarpa (wild). Figure 2 P. trichocarpa. heavily rusted. Figure 3 A l l poplars. P. trichocarpa. (A), P. 'Regenerata" (B) and P. 'Robusta' (C). Averages of both quarters. Figure 3. P. trichocarpa (nursery) Plate 20 ' Cross-sections of the wood in six-month old poplar shoots, taken near the base of the shoot. (X 80) Figure 1 P. trichocarpa Figure 2 P. 'Regenerata' Figure 3 P. 'Robusta' Plate 21 Cross-sections of the wood in six-month old poplar shoots, taken near the base of the shoot. (X 126) Figure 1 P. trichocarpa Figure 2 P. 'Regenerata' Figure 3 P. 'Robusta' Plate 22 Cross-sections of the bark in six-month old poplar shoots. (X 80) Figure 1 The upper shoot (quarter 2) of P. trichocarpa. Figure 2 The lower shoot (quarter U) of P. trichocarpa. Figure 3 The upper shoot (quarter 2) of P. 'Regenerata'. Figure 4 The lower shoot (quarter U) of P. 'Regenerata'. Plate 23 Cross-sections of the bark in six-month old poplar shoots. (X 80) Figure 1 The upper shoot (quarter 2) of P. 'Robusta1. Figure 2 The lower shoot (quarter 4-) of P. 'Robusta'. Plate 21+ Cross-sections of the periderm in six-month old poplar shoots, taken near the base of the shooto (X 126) Figure 1 P a trichocarpa Figure 2 P. 'Regenerata' Figure 3 P. 'Robusta' 

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