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Vesicular-arbuscular mycorrhizae and apples (Malus domestica Borkh.) in the nursery and in apple replant… Gamiet, Sharmin 1989

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VESICULAR-ARBUSCULAR MYCORRHIZAE AND APPLES (Malus domestica Borkh.) IN THE NURSERY AND IN APPLE REPLANT DISEASE by SHARMIN GAMIET B.Sc. Agr. (Hons), The University of B r i t i s h Columbia, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of S o i l Science) We accept t h i s thesis as conforming to the reguired standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1989 © S h a r m i n Gamiet, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT The purpose of t h i s study was t o determine i f d i f f e r e n t c l o n a l r o o t s t o c k from apple (Malus domestica Borkh.) formed d i f f e r e n t v e s i c u l a r a r b u s c u l a r m y c o r r h i z a l (VAM) a s s o c i a t i o n s . D i f f e r e n t f e r t i l i z e r s and VAM f u n g i were t e s t e d t o determine t h e i r e f f e c t s on apple s e e d l i n g growth i n apple r e p l a n t d i s e a s e d (ARD) s o i l s . VAM a s s o c i a t i o n s i n apple s t o o l b e d nursery were low. Over 80% of a l l samples had l e s s than 10% m y c o r r h i z a l c o l o n i z a t i o n . T h i s r e d u c t i o n i n VAM c o l o n i z a t i o n amongst v a r i o u s r o o t s t o c k c l o n e s i s a r e s u l t of d e t r i m e n t a l management p r a c t i c e s i n the s t o o l b e d n u r s e r y . Apples grown i n a budded n u r s e r y had h i g h m y c o r r h i z a l c o l o n i z a t i o n , the lowest c o l o n i z a t i o n r a t e was 30%. D i f f e r e n t r o o t s t o c k s from the budded nursery do not show any s i g n i f i c a n t d i f f e r e n c e s i n VAM c o l o n i z a t i o n , whereas c l o n a l r o o t s t o c k s from the s t o o l b e d nursery do. From the s t o o l b e d n u r s e r y , M a i l i n g (M) 2 c o n s i s t a n t l y showed h i g h e r VAM c o l o n i z a t i o n r a t e s , compared t o M 4, M 7 , M 9 , M 26, M a i l i n g Merton (MM) 106, MM 111, A l n a r p 2 and Ottawa 3. Apple r e p l a n t d i s e a s e (ARD) i s i d e n t i f i e d as the reason f o r poor growth o f apple s e d d l i n g s i n 5 s o i l s from the Okanagan V a l l e y o f B r i t i s h Columbia. S t e r i l i z a t i o n by a u t o c l a v i n g , p a s t e u r i z a t i o n and f o r m a l i n i n c r e a s e d t e s t s e e d l i n g h e i g h t . A i r -d r y i n g t e s t s o i l does not a f f e c t ARD pot b i o a s s a y s . However, a i r -d r y i n g the s o i l and p a s t e u r i z i n g or adding f o r m a l i n i n c r e a s e d p l a n t h e i g h t s i g n i f i c a n t l y more than these treatments i n n o n a i r -d r i e d s o i l s . The f e r t i l i z e r monoammonium phosphate (11-55-0) increased p l a n t height more than ammonium n i t r a t e (34-0-0) w h i l e t r i p l e superphosphate (0-45-0) d i d not increase p l a n t height. Root growth was increased by 0-45-0 only. VAM f u n g i were d r a s t i c a l l y reduced or e l i m i n a t e d by s t e r i l i z a t i o n and 11-55-0, but not by the other f e r t i l i z e r s . VAM f u n g i i n 2 ARD s o i l s do not overcome ARD. Test seedlings grown i n s t e r i l i z e d ARD s o i l s i n o c u l a t e d w i t h 4 species of VAM f u n g i do not show as great an increase i n shoot height compared to the a d d i t i o n of 11-55-0 f e r t i l i z e r . Root growth shows the i n v e r s e response. Glomus i n t r a r a d i c e s Schenck and Smith, was the best c o l o n i z e r but i n o c u l a t i o n w i t h G. versiforme (Karsten) Berch r e s u l t e d i n the g r e a t e s t shoot and root growth. Glomus clarum Nicholson and Schenck, and G. monosporum Gerdemann and Trappe, d i d not r e s u l t i n increases i n p l a n t growth i n ARD s o i l s . In s t e r i l i z e d ARD s o i l s , VAM f u n g i do not increase shoot growth as expected, but do increase root growth, suggesting the i n i t i a l growth of i n o c u l a t e d apple s e e d l i n g s i s root mass. Seedlings given 11-55-0 f e r t i l i z e r s show the reverse p a t t e r n of growth. In n o n s t e r i l i z e d ARD s o i l s , the growth of s e e d l i n g s appears to be i n v e r s e l y p r o p o r t i o n a l t o VAM c o l o n i z a t i o n . i i i TABLE OF CONTENTS page T i t l e Page i Abstract i i Table of Contents iv List of Tables v i i List of Figures x Acknowledgement x i i i CHAPTER 1:INTRODUCTION 1 CHAPTER 2: VA MYCORRHIZAE AND APPLES (Malus domestical IN THE NURSERY INTRODUCTION 11 MATERIALS AND METHODS 12 STOOLBED NURSERY 12 Mycorrhizal Status of Apple Rootstock 12 Distribution of VAM fungi 14 BUDDED NURSERY 2 0 Distribution of VAM fungi 2 0 COLD STORAGE AND ROOTSTOCK MYCORRHIZAE 2 3 VAM ANALYSIS 2 3 RESULTS 24 STOOLBEDS 2 4 Mycorrhizal Status of Apple Rootstock 24 Distribution of VAM Fungi 2 4 BUDDED NURSERY 3 2 Mycorrhizal Status of Apple Rootstock 32 Distribution of VAM Fungi 32 COLD STORAGE AND ROOTSTOCK MYCORRHIZAE 3 6 DISCUSSION 3 6 iv page CHAPTER 3: STERILIZING OLD APPLE SOILS INCREASES PLANT HEIGHT IN POT TESTS: CONFIRMATION OF THE PRESENCE OF APPLE REPLANT DISEASE INTRODUCTION 44 MATERIALS AND METHODS 4 6 COLLECTING SOILS FROM ORCHARDS 46 S o i l s 1 - 4 46 S o i l 5 48 POT BIOASSAY TEST FOR ARD 49 T e s t S e e d l i n g s 49 S o i l s 1 - 4 50 S o i l 5 52 VAM FUNGI 52 SOIL ANALYSIS 53 S o i l pH 53 S o i l Phosphorus 53 RESULTS 53 BIOASSAY TESTS 5 3 S e e d l i n g M o r t a l i t y 53 S o i l 1 - 4 53 Shoot Height 53 Root Dry Weight 54 VAM Fungi 54 S o i l A n a l y s i s 62 AIR-DRYING/ARD POT BIOASSAY 62 S o i l 5 62 Shoot Height 62 Root Dry Weight 62 VAM Fungi 69 S o i l A n a l y s i s 69 VAM FUNGI AND PLANT GROWTH 69 DISCUSSION 69 CHAPTER 4: APPLE GROWTH IN APPLE REPLANT DISEASED SOILS AFTER INOCULATION WITH VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGI INTRODUCTION 78 v page MATERIALS AND METHODS 81 THE SOILS 81 THE FUNGI 81 INOCULATING SOILS WITH VAM FUNGI 81 SEEDLING GROWTH AND ANALYSIS 82 RESULTS 82 SHOOT HEIGHT 82 SHOOT DRY WEIGHT 84 ROOT DRY WEIGHT 84 VAM FUNGI 89 VAM FUNGI AND PLANT GROWTH 89 DISCUSSION 93 CHAPTER 5: CONCLUSIONS 103 REFERENCES 110 APPENDIX 116 v i LIST OF TABLES CHAPTER 2 page 2.1 Rootstock M y c o r r h i z a l C o l o n i z a t i o n : A p r i l 1987 25 2.2 A n a l y s i s of V a r i a n c e f o r VAM i n Traas Nursery 28 2.3 A n a l y s i s o f V a r i a n c e f o r Mycorrhizae: Cannor Nursery 34 2.4 VAM C o l o n i z a t i o n Before and A f t e r C o l d Storage i n Rootstock from Stoolbeds 38 CHAPTER 3 3.1 S i t e L o c a t i o n and D e s c r i p t i o n o f Orchards 47 3.2 A n a l y i s of V a r i a n c e f o r Shoot Height: S o i l 1 55 3.3 A n a l y i s o f V a r i a n c e f o r Shoot Height: S o i l 2 56 3.4 A n a l y i s o f V a r i a n c e f o r Shoot Height: S o i l 3 57 3.5 A n a l y i s o f V a r i a n c e f o r Shoot Height: S o i l 4 58 3.6 A n a l y i s o f V a r i a n c e f o r Root Dry Weight: S o i l 1 . . . . 59 3.7 A n a l y i s o f V a r i a n c e f o r Root Dry Weight: S o i l 2 . . . . 60 3.8 A n a l y i s of V a r i a n c e f o r VAM C o l o n i z a t i o n : S o i l 1 . . . 61 3.9 A n a l y i s o f V a r i a n c e f o r VAM C o l o n i z a t i o n : S o i l 2 . . . 63 3.10a A n a l y s i s o f V a r i a n c e f o r S o i l pH: S o i l 1 64 3.10b A n a l y s i s o f V a r i a n c e f o r S o i l P: S o i l 1 64 3.10c S o i l pH and P (Olsen Method) Before and A f t e r S t e r i l i z a t i o n : S o i l 1 . . 64 3.11a A n a l y s i s o f V a r i a n c e f o r S o i l pH: S o i l 2 65 3.11b A n a l y s i s o f V a r i a n c e f o r S o i l P: S o i l 2 65 3.11c S o i l pH and P (Olsen Method) Before and A f t e r S t e r i l i z a t i o n : S o i l 2 65 J 3.12a A n a l y s i s o f V a r i a n c e f o r S o i l pH: S o i l 3 66 3.12b A n a l y s i s of V a r i a n c e f o r S o i l P: S o i l 3 66 v i i page 3.12c S o i l pH and P (Olsen Method) Before and A f t e r S t e r i l i z a t i o n : S o i l 3 66 3.13a A n a l y s i s o f V a r i a n c e f o r S o i l pH: S o i l 4 67 3.13b A n a l y s i s o f V a r i a n c e f o r S o i l P: S o i l 4 67 3.13c S o i l pH and P (Olsen Method) Before and A f t e r S t e r i l i z a t i o n : S o i l 4 67 3.14 A n a l y s i s o f V a r i a n c e f o r Shoot Height: S o i l 5 68 3.15 A n a l y s i s o f V a r i a n c e f o r Root Dry Weight: S o i l 5 . . . 70 3.16a A n a l y s i s o f V a r i a n c e f o r Mycorrhizae: S o i l 5 71 3.16b Percent M y c o r r h i z a l C o l o n i z a t i o n : S o i l 5 71 3.17 A n a l y s i s o f V a r i a n c e f o r S o i l pH: S o i l 5 72 3.18 A n a l y s i s o f V a r i a n c e f o r S o i l P: S o i l 5 73 CHAPTER 4 4.1 A n a l y s i s of V a r i a n c e f o r Shoot Height: S o i l 2 83 4.2 A n a l y s i s of V a r i a n c e f o r Shoot Height: S o i l 5 85 4.3 A n a l y s i s o f V a r i a n c e f o r Shoot Dry Weight: S o i l 2 .. 86 4.4 A n a l y s i s o f V a r i a n c e f o r Shoot Dry Weight: S o i l 5 .. 87 4.5 A n a l y s i s o f V a r i a n c e f o r Root Dry Weight: S o i l 2 . . . 88 4.6 A n a l y s i s o f V a r i a n c e f o r Root Dry Weight: S o i l 5 . . . 90 4.7 A n a l y s i s o f V a r i a n c e f o r Mycorrhizae: S o i l 2 91 4.8 A n a l y s i s o f V a r i a n c e f o r Mycorrhizae: S o i l 5 92 v i i i LIST OF FIGURES INTRODUCTION page 1.1 Apple Propagation i n Stoolbeds 2 1.2 Apple Stoolbeds i n S o i l 3 1.3 Apples i n Budded Nursery 3 1.4 Low Density Orchard 5 1.5 High Density Orchard 5 1.6 Apple T r a n s p l a n t Showing Apple Replant Disease 6 1.7 Mycorrhizae i n Apples 9 CHAPTER 2 2.1 Sampling S t r a t e g i e s f o r Traas Nursery: F i e l d s 1 and 2 15 2.2 Sampling S t r a t e g i e s f o r Traas Nursery: F i e l d 3 16 2.3 Sampling S t r a t e g i e s f o r Traas Nursery: F i e l d s 4 17 2.4 Schematic Diagram of a Sample 19 2.5 C o l l e c t i n g Roots from Sub-Sample S i t e 19 2.6 Sampling S t r a t e g i e s f o r Cannor Nursery: F i e l d 1 21 2.7 Sampling S t r a t e g i e s f o r Cannor Nursery: F i e l d 2 22 2.8 Mean and Range f o r Percent C o l o n i z a t i o n i n Nine D i f f e r e n t Rootstocks from Stoolbeds: A p r i l 1987 25 2.9 VAM D i s t r i b u t i o n i n Stoolbeds: F a l l 1987 27 2.10 Rootstock C o l o n i z a t i o n , Traas Nursery: F a l l 1987 . . . . 28 2.11 P a t t e r n s of VAM i n Stoolbeds: Traas Nursery F i e l d 1 . 29 2.12 P a t t e r n s of VAM i n Stoolbeds: Traas Nursery F i e l d 2 . 30 2.13 P a t t e r n s of VAM i n Stoolbeds: Traas Nursery F i e l d 3 . 31 i x page 2.14 P a t t e r n s o f VAM i n Stoolbeds: Traas Nursery F i e l d 4 . 33 2.15 M y c o r r h i z a l C o l o n i z a t i o n i n Cannor Nursery 34 2.16 P a t t e r n s of VA Mycorrhizae: Cannor Nursery F i e l d 1 .. 35 2.17 P a t t e r n s of VA Mycorrhizae: Cannor Nursery F i e l d 2 .. 37 CHAPTER 3 3.1 P l a n t Growth Bench 51 3.2 Mean Shoot Height a t 10 Weeks: S o i l 1 55 3.3 Mean Shoot Height at 10 Weeks: S o i l 2 56 3.4 Mean Shoot Height at 10 Weeks: S o i l 3 57 3.5 Mean Shoot Height at 10 Weeks: S o i l 4 58 3.6 Mean Root Dry Weight at 10 Weeks: S o i l 1 59 3.7 Mean Root Dry Weight at 10 Weeks: S o i l 2 60 3.8 Percent M y c o r r h i z a l C o l o n i z a t i o n : S o i l 1 61 3.9 Percent M y c o r r h i z a l C o l o n i z a t i o n : S o i l 2 63 3.10 Mean Shoot Height: S o i l 5 68 3.11 Mean Root Dry Weight: S o i l 5 70 3.12 S o i l pH: S o i l 5 72 3.13 S o i l Phosphorus (Olsen Method): S o i l 5 73 3.14 P l a n t Height and VA M y c o r r h i z a l C o l o n i z a t i o n : S o i l 1 74 3.15 P l a n t Height and VA M y c o r r h i z a l C o l o n i z a t i o n : S o i l 2 74 3.16 P l a n t Height and VA M y c o r r h i z a l C o l o n i z a t i o n : S o i l 5 74 CHAPTER 4 4.1 Shoot Height a t 10 Weeks: S o i l 2 83 4.2 Shoot Height at 10 Weeks: S o i l 5 85 4.3 Shoot Dry Weight a t 10 Weeks: S o i l 2 86 x page 4.4 Shoot Dry Weight a t 10 Weeks: S o i l 5 87 4.5 Root Dry Weight at 10 Weeks: S o i l 2 88 4.6 Root Dry Weight at 10 Weeks: S o i l 5 90 4.7 Percent M y c o r r h i z a l C o l o n i z a t i o n : S o i l 2 91 4.8 Percent M y c o r r h i z a l C o l o n i z a t i o n : S o i l 5 92 4.9 M y c o r r h i z a l C o l o n i z a t i o n and P l a n t Height: S o i l 2 . . . 94 4.10 M y c o r r h i z a l C o l o n i z a t i o n and P l a n t Height: S o i l 5 . . . 95 4.11 M y c o r r h i z a l C o l o n i z a t i o n and Root Dry Weight: S o i l 2 96 4.12 M y c o r r h i z a l C o l o n i z a t i o n and Root Dry Weight: S o i l 5 97 x i ACKNOWLEDGEMENTS I wish t o thank the Science C o u n c i l o f B r i t i s h Columbia and the B.C. F r u i t Growers A s s o c i a t i o n f o r funding t h i s p r o j e c t . I wish t o thank Drs G. H. N i e l s o n , A.A. Bomke and L.M. L a v u k u l i c h f o r t h e i r h e l p f u l comments i n the w r i t e up of t h i s t h e s i s . My s i n c e r e s t g r a t i t u d e t o Dr. M. G r e i g f o r the hours spent wi t h me i n t r y i n g t o make sense of and then h e l p i n g me analyze the data of t h i s t h e s i s . To my c o l l e g u e s , Barbara Cade, B i l l Chapman, E l i s a b e t h Deom and Aaron Roth, I g i v e a h e a r t f u l thanks f o r the he l p and e s p e c i a l l y f o r a l l the ideas t h a t flowed so abundantly. To Dr. S.M. Berch, I am e t e r n a l l y indebted f o r the p a t i e n c e , a d v i c e , h e l p , guidance, encouragement and e s p e c i a l l y f o r the c o n v i c t i o n she had i n me. Without her s u p e r v i s i o n t h i s t h e s i s c o u l d not have been completed. M o s t l y , t o my f a m i l y , F r e d , Riyad and F a r i d a R e i d , who b e w i l d e r e d l y l i s t e n e d t o the wonders of r o o t s and f u n g i and who were s u p p o r t i v e throughout t h i s time I say 'Modassi Paa and i t s over'. x i i INTRODUCTION Apples (Malus domestica Borkh) are i n i t i a l l y propagated i n stoolbeds. P r i o r t o e s t a b l i s h i n g the stoolbed, the s o i l i s fumi g a t e d and fu n g i c i d e s are a p p l i e d f o r disease c o n t r o l throughout the growing season. A mother p l a n t i s e s t a b l i s h e d and allowed t o grow f o r one season (Figure 1.1). In the f a l l , a f t e r l e a f drop, the mother p l a n t i s cut j u s t above the s o i l l i n e . The f o l l o w i n g s p r i n g new shoots a r i s e from the o l d mother p l a n t . Throughout the growing season s o i l or sawdust i s h i l l e d around the new shoots to i n i t i a t e r o o t i n g (Figure 1.2). In the f a l l , a f t e r the buds are dormant, the rooted shoots (rootstock) are cut j u s t above the s o i l l i n e l e a v i n g the roo t s of the mother p l a n t i n the stoolbed. The mother p l a n t overwinters and the f o l l o w i n g s p r i n g , the c y c l e begins again. The r o o t s t o c k s are placed i n c o l d storage u n t i l the f o l l o w i n g s p r i n g when they are shipped t o the orchard or to another nursery f o r budding (Figure 1.3). In the budded nursery the r o o t s t o c k s are planted i n the s p r i n g and are allowed t o grow v i g o r o u s l y t i l l mid summer. In the l a t t e r p a r t of August, the shoots are budded about 15 cm above the s o i l l i n e . The ro o t s t o c k w i t h i t s new bud overwinters i n the nursery. In the s p r i n g , the o r i g i n a l shoots of the roo t s t o c k are cut j u s t above the bud union. The buds are allowed t o grow f o r 1 season and the roo t s t o c k w i t h i t s new s c i o n i s ready f o r harvest i n the f a l l . A f t e r 2 growing seasons i n the budded nursery, the roots t o c k 1 Stool bed started by planting a Mother plant grows for one Top is removed to 1 in. above rooted layer in a small trench, season to become established, ground just before growth begins. When new shoots are 3 fo 5 in. high soil or sawdust is added to half their height. Soil is then added at intervals until it is 6 to 8 in. deep. At end of season roots have formed at base of covered shoots. Rooted layers are cut off as closely as possible to the base and are lined out in nursery row. Mother stool with loyers removed at the beginning of the next season. Additional new shoots will be layered. Figure i . l : Apple propagation i n Stoolbeds (Hartman, H.T. and D.E. Kester 1975) 2 F i g u r e 1.2: A p p l e S t o o l b e d s i n S o i l and i t s s c i o n are l i f t e d , p l a c e d i n c o l d s t o r a g e and shipped to the o r c h a r d . C u l t i v a t i o n of apples i n the o r c h a r d i s changing. Growers are moving away from the low d e n s i t y , v i g o r o u s t r e e s , t o the h i g h d e n s i t y , dwarfing t r e e s ( F i g u r e s 1.4 and 1.5). The i n i t i a l c o s t of e s t a b l i s h i n g these i n t e n s i v e orchards i s h i g h , and the grower must be a b l e t o ensure a r e t u r n on investments as soon as p o s s i b l e . The orchards must t h e r e f o r e be i n f u l l p r o d u c t i o n w i t h i n 3 - 4 y e a r s . Any f a c t o r s which prevent h e a l t h y , e a r l y p r o ducing orchards must be a v o i d e d . Apple r o o t s t o c k t r a n s p l a n t e d i n t o o l d apple o r c h a r d s o i l w i l l o f t e n not grow ve r y w e l l . In the f i r s t year i n the orchard the s c i o n does not e l o n g a t e , t h e r e i s l i t t l e i n t e r n o d e growth and t h e r e i s no f i b r o u s r o o t growth (F i g u r e 1.6). No one pathogen i s always a s s o c i a t e d w i t h t h i s d i s o r d e r . However, i f the orchard s o i l i s fumigated p r i o r t o t r a n s p l a n t i n g the s c i o n w i l l o f t e n grow more v i g o r o u s l y . T h i s d i s o r d e r i s known as a ' r e p l a n t d i s e a s e * . T h i s d i s e a s e i s p r e f i x e d by the host i t i s a s s o c i a t e d w i t h . Thus t h i s d i s e a s e a s s o c i a t e d w i t h apples i s r e f e r r e d t o as apple r e p l a n t d i s e a s e (ARD). ARD has been r e p o r t e d f o r over 300 years and i s found i n a l l apple growing areas of the world (Buszard and Jensen 1986, Hoestra 1968, Ryan 1975, Mai and Abawi 1981, Ross et a l . 1984, Benson e t a l . 1978, Savory 1967, S l y k h u i s and L i 1985). Replant d i s e a s e s are found i n numerous p l a n t s , e s p e c i a l l y f r u i t and p l a n t a t i o n crops (Deal et a l . 1971, Havis et a l . 1958, Hwang 4 Figure 1.4: Low Density Orchard F i g u r e 1.6: A p p l e T r a n s p l a n t Showing A p p l e R e p l a n t D i s e a s e a) S t u n t e d Shoot Growth b) H e a l t h y Tree c) S h o r t e n e d I n t e r n o d e s d) Reduced Root System 6 r 1988, Mountain and Boyce 1958, Pepin e t a l . 1975, T r u d g i l l 1984). No s i n g l e pathogen has been i d e n t i f i e d w i t h a l l r e p o r t e d cases of ARD. In C z e c h o s l a v a k i a , the f l u o r e s c e n t pseudomonads (Catska e t a l . 1982) and P e n i c i l l i u m c l a v i f o r m e (Catska e t a l . 1988) have been i d e n t i f i e d as the c a u s a l agents of ARD. The fungus Pythium spp. i n Great B r i t a i n , (Sewell 1981) and nematodes i n H o l l a n d (Hoestra and Oostenbrink 1962) and New York ( J a f f e et a l . 1982) have a l s o been i d e n t i f i e d as the c a u s a l agents of ARD. I t t h e r e f o r e appears t h a t though a c o n t r i b u t i n g agent i s b i o l o g i c a l , i t i s not a simple d i s e a s e . S l y k h u i s and L i (1985) and Sewell and Roberts (1985) r e p o r t e d t h a t i n a d d i t i o n t o f u m i g a t i o n , ARD i s overcome by the a d d i t i o n of h i g h n i t r o g e n and phosphorus f e r t i l i z e r s . The importance of phosphorus i n apple growth i s w e l l documented ( M i l l e r e t a l . 1985b, Hoepfner et a l . 1983) . The b i o l o g i c a l n a ture of the d i s e a s e and the phosphorus f a c t o r suggest t h a t mycorrhizae may p l a y a r o l e i n ARD. Apples form a m u t u a l i s t i c a s s o c i a t i o n w i t h v e s i c u l a r -a r b u s c u l a r m y c o r r h i z a l (VAM) f u n g i . The fungus a c t s as r o o t e x t e n s i o n s e x p l o r i n g s o i l t h a t i s i n a c c e s s i b l e t o the p l a n t , moving phosphorus from the s o i l m a t r i x through the hyphae and i n t o the p l a n t . At s i t e s w i t h i n the c e l l , the fungus exchanges phosphorus f o r simple sugars from i t s host p l a n t . The fungus forms i n t e r - and i n t r a c e l l u l a r hyphae i n the p l a n t r o o t . A f t e r p e n e t r a t i o n of the r o o t e p i d e r m i s , the hyphae c o i l and spread throughout the c o r t e x of the r o o t but never enter 7 the meristem t i s s u e ( F i g u r e 1.7a). Hyphae p e n e t r a t e the c o r t i c a l c e l l s then r e p e a t e d l y branch dichotomously. These branches take on the form of ' l i t t l e t r e e s * and are known as a r b u s c u l e s (Figure 1.7b). N u t r i e n t t r a n s f e r between the host and r o o t occur w i t h i n t h e s e a r b u s c u l e s . During the l a t e r stages of development, v e s i c l e s form i n t e r c e l l u l a r l y and are f i l l e d w i t h o i l d r o p l e t s ( F i g u r e 1.7c). Hyphae grow o u t s i d e the r o o t and may t e r m i n a t e i n chlamydospores, though these spores may a l s o be found w i t h i n the r o o t . I d e n t i f i c a t i o n o f the fungus i s based on spore morphology. Both the v e s i c l e s and spores are r e s i s t a n t t o harsh environmental c o n d i t i o n s . N u r s e r i e s are i r r i g a t e d r e g u l a r l y , g i v e n adequate amounts of f e r t i l i z e r s and h e a v i l y sprayed w i t h p e s t i c i d e s . I f VAM f u n g i are found i n n u r s e r y beds, they w i l l be adapted t o t h a t environment. I f r o o t s t o c k s are m y c o r r h i z a l upon l i f t i n g from the n u r s e r y , the mycorrhizae may not s u r v i v e c o l d s t o r a g e . Old a pple orchards have p o p u l a t i o n s o f VAM f u n g i t h a t have s t a b i l i z e d over the y e a r s . New apple r o o t s t o c k t r a n s p l a n t e d i n t o these o l d apple s o i l s may not b e n e f i t from mycorrhizae indigenous to o l d apple s o i l s . The purpose of t h i s study was t o : 1) determine the m y c o r r h i z a l s t a t u s of apple r o o t s t o c k grown i n s t o o l b e d s ; 2) e s t a b l i s h the d i s t r i b u t i o n o f m y c o r r h i z a l f u n g i i n s t o o l b e d and budding n u r s e r i e s ; 3) determine the e f f e c t s o f c o l d s torage on the mycorrhizae of d i f f e r e n t r o o t s t o c k s ; 4) i d e n t i f y d i f f e r e n t ARD s o i l s from the Okanagan V a l l e y of B r i t i s h Columbia; 5) 8 Figure 1.7: Mycorrhizae i n Apples a) Root p e n e t r a t i o n and hyphal spread b) Arbuscules w i t h i n c o r t i c a l c e l l s c) V e s i c l e s d) Stained e x t e r n a l spores 9 determine the e f f e c t s of v a r i o u s f e r t i l i z e r s on growth of apple s e e d l i n g s i n ARD pot b i o a s s a y s ; 6) determine i f a i r - d r y i n g ARD s o i l s a f f e c t s ARD pot b i o a s s a y s ; and 7) determine the e f f e c t s of i n o c u l a t i n g ARD s o i l s with v a r i o u s VAM f u n g i on the growth of apple s e e d l i n g s i n pot t e s t s . 10 VA MYCORRHIZAE AND APPLES (Malus domestical IN THE NURSERY INTRODUCTION There i s i n c r e a s i n g i n t e r e s t i n the r o l e of mycorrhizae i n many orcha r d problems. Apples t h a t have problems becoming e s t a b l i s h e d i n orchards and t h a t respond t o the a d d i t i o n s of phosphorus f e r t i l i z e r s may have no VAM f u n g i , o r may have VAM f u n g i t h a t are not adapted t o the o r c h a r d environment. D i f f e r e n t c l o n a l apple r o o t s t o c k i n the o r c h a r d have been shown t o have d i f f e r e n t m y c o r r h i z a l f u n g i ( M i l l e r e t a l . 1985a), though t h i s d i f f e r e n c e , was thought t o be a r e s u l t of the geographic d i s t r i b u t i o n o f the orchard r a t h e r than the r o o t s t o c k t y p e . The m y c o r r h i z a l s t a t u s of apple r o o t s t o c k p r i o r t o t r a n s p l a n t i n g i s important and should be e s t a b l i s h e d . Management p r a c t i c e s i n the nursery may a d v e r s e l y a f f e c t VAM f u n g i . C e r t i f i e d strawberry p l a n t s grown i n nursery f i e l d s fumigated p r i o r t o e s t a b l i s h m e n t showed low VAM c o l o n i z a t i o n i n t e n s i t i e s d u r i n g the f i r s t year of growth but i n the second yea r showed a wide range of c o l o n i z a t i o n (Robertson e t a l . 1988). Apples are propagated i n nursery s t o o l b e d s , h a r v e s t e d , p l a c e d i n c o l d s t o r a g e and then t r a n s p l a n t e d i n t o another nursery where they are budded. P r i o r t o e s t a b l i s h i n g the s t o o l b e d , the s o i l i s fumigated. In c i t r u s and peach n u r s e r i e s , p l a n t s grown i n fumigated s o i l showed n u t r i e n t d e f i c i e n c i e s l i n k e d t o the absence of VAM f u n g i (Kleinschmidt and Gerdemann 1978, La Rue et a l . 1975). I n o c u l a t i n g these n u r s e r i e s w i t h VAM f u n g i overcame t h i s problem. 11 While apples i n st o o l b e d s may not show n u t r i e n t d e f i c i e n c i e s i n fumigated s o i l s , they are h e a v i l y f e r t i l i z e d and giv e n numerous a p p l i c a t i o n s o f f u n g i c i d e throughout the season. These p r a c t i c e s are most l i k e l y t o be d e t r i m e n t a l t o VAM f u n g i i n s t o o l b e d s . I t i s w e l l documented t h a t i n the presence o f h i g h P, VAM f u n g i are suppressed (Hoepfner e t a l . 1983, M i l l e r e t a l . 1985b). I f m y c o r r h i z a l f u n g i do become e s t a b l i s h e d i n nursery s t o o l b e d s , i t i s p o s s i b l e t h a t d i f f e r e n t c l o n a l r o o t s t o c k s may form d i f f e r e n t VAM a s s o c i a t i o n s . There i s no i n f o r m a t i o n a v a i l a b l e on the mycorrhizae present i n c l o n a l r o o t s t o c k from n u r s e r i e s . I f mycorrhizae are p r e s e n t , the d i s t r i b u t i o n of the f u n g i throughout the nursery would be a major f a c t o r on the p r o b a b i l i t y o f a r o o t s t o c k becoming c o l o n i z e d b e f o r e l e a v i n g the n u r s e r y . The o b j e c t i v e s o f t h i s study were t o : 1) e s t a b l i s h the m y c o r r h i z a l s t a t u s of d i f f e r e n t c l o n a l r o o t s t o c k from s t o o l b e d s from the F r a s e r V a l l e y o f B.C.; 2) determine the d i s t r i b u t i o n o f VAM f u n g i i n s t o o l b e d and budded n u r s e r i e s i n the F r a s e r V a l l e y of B.C.; 3) t o determine the e f f e c t s of c o l d s t o r a g e on the mycorrhizae i n d i f f e r e n t r o o t s t o c k from a s t o o l b e d nursery from the F r a s e r V a l l e y of B.C. MATERIALS AND METHODS STOOLBED NURSERY Mycorrhizal Status of Apple Rootstock Rootstock sampling o c c u r r e d a t Traas Nursery, 24355 - 48th Avenue, Langley, B.C. The s o i l belongs t o Marble H i l l s e r i e s 12 which has over 50 cm of medium t e x t u r e d a e o l i a n d e p o s i t s over g r a v e l l y g l a c i a l outwash d e p o s i t s . I t i s a w e l l d r a i n e d ortho h u m o - f e r r i c podzol w i t h 0.5 - 2.0% s l o p e (Luttmerding 1980). In A p r i l 1987, 9 d i f f e r e n t apple r o o t s t o c k s , propagated i n s t o o l beds i n s o i l , were examined f o r the presence of VAM f u n g i . Two sampling s t r a t e g i e s were used f o r t h i s s t u d y . M a i l i n g (M) 4, M 7a, and M a i l i n g Merton (MM) 111, from the e n t i r e n u r s ery had a l r e a d y been l i f t e d and were e i t h e r i n c o l d s t o r a g e or being processed f o r c o l d s t o r a g e . Ten t r e e s from each r o o t s t o c k were taken randomly from the storage p i l e and then p l a c e d i n c o l d s t o r a g e , s e p a r a t e from the commercial s t o c k . MM 106, M 2, M 7, M 26, A l n a r p 2 and Ottawa 3 were s t i l l i n the f i e l d . From each r o o t s t o c k b l o c k , 10 t r e e s were tagged randomly w i t h f l u o r e s c e n t surveyor t a p e . Through the f a l l and w i n t e r , 1986/87, as each r o o t s t o c k went dormant and was l i f t e d , tagged t r e e s were p l a c e d i n c o l d storage separate from the commercial s t o c k . T h r o u g h o u t t h e w i n t e r , t a g g e d t r e e s were w a t e r e d o c c a s i o n a l l y t o prevent d e s s i c a t i o n of the r o o t s . In A p r i l 1987, a l l the r o o t s t o c k had been l i f t e d and the r o o t s from each sampled t r e e were p l a c e d i n FAA (Formaldehyde (90) : A c e t i c A c i d (5) : E t h a n o l ( 5 ) ) , ( v o l : v o l : v o l : ) and examined f o r the presence of VAM. A l l samples taken showed a b i n o m i a l d i s t r i b u t i o n and were t h e r e f o r e a n a l y s e d u s i n g the Mann-Whitney U non-parametric t e s t f o r s i g n i f i c a n t d i f f e r e n c e s i n percent c o l o n i z a t i o n . 13 D i s t r i b u t i o n of VAM Fungi In the f a l l o f 1987, Traas Nursery was sampled i n g r e a t e r d e t a i l , i n o r d e r t o determine the d i s t r i b u t i o n o f VAM f u n g i i n the f i e l d s . The r o o t s t o c k s sampled were i d e n t i c a l t o those used above but M 9 was i n c l u d e d i n t h i s s tudy. Four f i e l d s , each c o n t a i n i n g d i f f e r e n t combinations of r o o t s t o c k were sampled. The m a t u r i t y o f the s t o o l b e d s v a r i e d , the o l d e s t one i s i n f i e l d 4 and i s 18 y e a r s o l d , the youngest i s an M 9 b l o c k i n f i e l d 3 which i s o n l y 2 years o l d . A l l r o o t s t o c k s were not n e c e s s a r i l y found i n each f i e l d . Each s t o o l b e d was fumigated p r i o r t o e s t a b l i s h m e n t , and then f e r t i l i z e d and g i v e n p e s t i c i d e s throughout the year as necessary. To h e l p i n i t i a t e r o o t i n g , p i n e bark mulch was spread on the rows and then s o i l was h i l l e d around the growing s h o o t s . Each f i e l d was blocked a c c o r d i n g t o r o o t s t o c k ( F i g u r e 2.1, 2.2 and 2.3). Number of b l o c k s per f i e l d v a r i e d depending on the p o s i t i o n and number of rows i n each b l o c k . The number of samples taken from each b l o c k v a r i e d a c c o r d i n g t o the s i z e o f the b l o c k . The minimum number of samples taken from a b l o c k was 1 and the maximum was 12 ( F i g u r e s 2.1, 2.2, 2.3). The area of each of the l a r g e r b l o c k s was d i v i d e d i n t o 100 equal s e c t i o n s . Lotus 123 software computer package was used t o generate 2 random numbers each used t o i n d i c a t e a p o s i t i o n on the v e r t i c a l and h o r i z o n t a l a x i s . The sample p o s i t i o n w i t h i n a b l o c k was l o c a t e d a t the i n t e r s e c t o f these 2 axes. Each sample was composed of of 3 sub-samples from which 14 Figure 2.2: Sampling Strategies for Traas Nursery: F i e l d 3 16 Figure 2.3: Sampling Strategies for Traas Nursery: F i e l d 4 17 r o o t s were c o l l e c t e d . A sample was 20 meters long and was d i v i d e d i n t o 3 sub-samples of which two, (sub-samples 2 and 3) were l o c a t e d 8.3 m from a c e n t r a l sub-sample (sub-sample 1) (Figure 2.4) . At each sub-sample s i t e the s o i l was brushed a s i d e t o expose the r o o t s o f each r o o t s t o c k . The beds had been kept weed-free throughout the growing season and the r o o t s of the young t r e e s were s u b e r i z e d a l r e a d y and were t h e r e f o r e easy t o i d e n t i f y ( F i g u r e 2.5b). The r o o t s were sh a l l o w , so i t was not necessary t o d i g deeper than 15 cm t o expose them ( F i g u r e 2.5a). Three r o o t s , at l e a s t 3 mm - 6 mm i n diameter with approximately 50 l a t e r a l s , were each removed from the a p i c a l p o r t i o n of the r o o t system and b u l k e d . The e x c i s e d r o o t s were p l a c e d i n a p o l y e t h y l e n e bag w i t h about 300 mis of s o i l . Those r o o t s s t i l l a t t a c h e d t o the young t r e e s were then re-covered with s o i l . A l l bags c o n t a i n i n g r o o t s and s o i l were brought t o the l a b o r a t o r y where the r o o t s were p l a c e d i n l a b e l l e d b o t t l e s and f i x e d i n FAA. The s o i l was a i r - d r i e d and then s t o r e d . A l l r o o t s were examined f o r VAM f u n g i . Sampling o c c u r r e d f i e l d by f i e l d over 5 days. F i e l d 2 was sampled f i r s t , f o l l o w e d by F i e l d s 1, 3, and 4. The data c o l l e c t e d f o r a l l f o u r f i e l d s were a r c s i n e transformed (Zar 1984) p r i o r t o s t a t i s t i c a l a n a l y s i s . A m o d i f i e d Nested Two Way A n a l y s i s of V a r i a n c e was used t o analyze the d a t a . Tukey's m u l t i p l e range t e s t was used t o t e s t f o r any d i f f e r e n c e s amongst the f i e l d s , r o o t s t o c k s , b l o c k s and any i n t e r a c t i o n s 18 Figure 2.5: a) C o l l e c t i n g Roots from Sub-Sample S i t e ; b ) . Apple Roots from Stoolbed 19 amongst these v a r i a b l e s . BUDDED NURSERY D i s t r i b u t i o n o f VAM Fungi During the f i r s t and second week of November, 1987, two f i e l d s from Cannor N u r s e r i e s a t the Froese s i t e , 10020 G i l l a n d e r s Road, C h i l l i w a c k , B.C. were sampled t o determine the d i s t r i b u t i o n of VAM f u n g i i n a budded n u r s e r y . T h i s s o i l belongs t o the Monroe S e r i e s which i s medium-textured wi t h a s i l t y loam t e x t u r e and has l a t e r a l l y a c c r e t e d f l o o d p l a i n d e p o s i t s . I t i s moderately w e l l t o w e l l d r a i n e d . T h i s s o i l has a shallow phase which means t h a t t h e r e are areas of the s o i l t h a t are s i m i l a r i n a l l r e s p e c t s to t h e named s o i l , except t h a t the depth of the s o i l p r o f i l e i s s h a l l o w e r than the modal. I t has a 3% s l o p e and i s c l a s s i f i e d as an e l u v i a t e d e u t r i c b r u n i s o l (Luttmerding 1981). The s o i l i n t h i s n u r s e ry i s not fumigated p r i o r t o t r a n s p l a n t i n g r o o t s t o c k , and only sprayed 1 - 2 times w i t h a copper c h l o r i d e f u n g i c i d e per season f o r d i s e a s e c o n t r o l . A l l r o o t s t o c k s had spent 2 seasons i n the f i e l d and were t o be shipped i n the s p r i n g of 1988. A number of the r o o t s t o c k s examined from t h i s nursery o r i g i n a t e d from Traas N ursery. Each r o o t s t o c k had been budded wit h d i f f e r e n t s c i o n v a r i e t i e s . M a i l i n g 27 was the o n l y r o o t s t o c k found i n t h i s n u r s e ry t h a t was not a l s o found i n the s t o o l b e d n u r s e r y . Rootstocks examined were M 4, M 7, M 9, M 26, M 27 and A l n a r p 2. Sampling s t r a t e g i e s f o r t h i s nursery were i d e n t i c a l t o those of the s t o o l b e d nursery ( F i g u r e s 2.6 and 2.7). 20 Figure 2.6: Sampling Strategies for Cannor Nursery: F i e l d 1 2 1 Figure 2.7: Sampling S t r a t e g i e s f o r Cannor Nursery: F i e l d 2 22 A m o d i f i e d nested ANOVA was used t o t e s t f o r s i g n i f i c a n t d i f f e r e n c e s between the 2 f i e l d s and r o o t s t o c k s . No i n t e r a c t i o n between the f i e l d s and r o o t s t o c k s was p o s s i b l e as M 26 was the on l y r o o t s t o c k o f the 6 examined t h a t o c c u r r e d i n both f i e l d s . COLD STORAGE AND ROOTSTOCK MYCORRHIZAE Rootstocks MM 111, MM 106, M 26, M 4 and M 7 from Traas Nursery were used i n t h i s s tudy. MM 111 and M 7 were l i f t e d from the f i e l d over the p e r i o d of 15 November t o December, 1 1987 and were p l a c e d i n c o l d s torage (2-4°C and 75-80% humidity) on 4th of January, 1988. On January 5, 1988, 10 p l a n t s were taken randomly from each o f the st o r a g e p i l e s o f these 2 r o o t s t o c k s . The e n t i r e r o o t system from each p l a n t were f i x e d i n FAA. By January 9, 1988, MM 106, 1 f i e l d o f M 26, and M 4 had a l s o been l i f t e d though they were not processed f o r c o l d s torage y e t . Ten p l a n t s from these 3 r o o t s t o c k s were taken from the sto r a g e p i l e and t h e i r r o o t s f i x e d i n FAA, f o r VAM counts. A f t e r spending 15 weeks i n c o l d s torage a t 2 - 4°C and r e l a t i v e humidity o f 75 - 80% they were again sampled. M 4 had not been p l a c e d i n c o l d s t o r a g e , but r a t h e r i n a d r y , dark, c o o l shed. Ten p l a n t s from each o f the r o o t s t o c k p i l e s were taken. The r o o t s from each of the t r e e s were f i x e d i n FAA f o r VAM counts. VAM ANALYSIS A l l r o o t s were examined f o r the presence o f mycorrhizae u s i n g a m o d i f i e d v e r s i o n o f Kormanik and McGraw's (1982) t e c h n i q u e . A l l r o o t s were d a r k l y s t a i n e d , so i t was necessary t o b l e a c h r o o t s f o r 20 - 30 minutes i n 30% a l k a l i n e H202. Percent m y c o r r h i z a l c o l o n i z a t i o n was measured u s i n g the g r i d l i n e i n t e r s e c t method ( G i o v a n e t t i and Mosse 1980). S u r f e r , a computer software package was used t o prepare a l l VAM f u n g a l d i s t r i b u t i o n graphs. RESULTS STOOLBEDS Mycorrhizal Status of Apple Rootstocks V e s i c l e s and hyphae were the o n l y VAM f u n g a l s t r u c t u r e s observed i n the r o o t s of a l l samples examined. S i g n i f i c a n t d i f f e r e n c e s i n percent c o l o n i z a t i o n amongst the nine r o o t s t o c k s examined were obt a i n e d a t 0.05 c o n f i d e n c e l i m i t s (Table 2.1). R e s u l t s o b t a i n e d i n t h i s study show a l a r g e v a r i a t i o n i n p e r c e n t c o l o n i z a t i o n w i t h i n and amongst the d i f f e r e n t r o o t s t o c k s ( F i g u r e 2.8). Only M 2 had mycorrhizae p r e s e n t i n a l l 10 samples examined. M a i l i n g 4, M 7 and MM 111 had 2 of the 10 samples examined wi t h 0% c o l o n i z a t i o n . A l n a r p 2, and M 7a had 3 p l a n t s of the 10 examined wi t h 0 % c o l o n i z a t i o n w h i l e Ottawa 3, M 26 and MM 106 had 4, 5, and 6 samples r e s p e c t i v e l y w i t h 0% c o l o n i z a t i o n . M a i l i n g 2 had a low of 10% VAM c o l o n i z a t i o n and a h i g h of 80%. The remaining r o o t s t o c k s had highs of under 50%. M a i l i n g 2 had the h i g h e s t mean at 37%, f o l l o w e d by M 7 at 32%. M a i l i n g Merton 106 and A l n a r p 2 had the lowest mean percent c o l o n i z a t i o n a t 2% and 9% r e s p e c t i v e l y (Table 2.1). D i s t r i b u t i o n of VAM Fungi Mycorrhizae were found i n p l a n t s from a l l 4 f i e l d s . However, the c o l o n i z a t i o n r a t e i n a l l f i e l d s was g e n e r a l l y low. F i f t y 24 c o O N ' c _o o o c CD O k_ Q_ 1 0 0 n 3 5 0 -0 MM 111 M 4 M 7a Ottawa 3 MM 106 M 2 M 7 M 26 Alnarp 2 ROOTSTOCK F i g u r e 2.8: Mean and Range f o r C o l o n i z a t i o n o f Nine D i f f e r e n t Rootstocks from St o o l b e d s : A p r i l 1987 Rootstock % Mean C o l o n i z a t i o n MM 106 2 a* A l n a r p 2 9 a,b Ottawa 3 13 a, c M 7a 14 a,d MM 111 14 b,c,d,e M 26 15 a,e, f M 4 18 b,c,d,f M 7 32 c,d , f , g M 2 37 g Tabl e 2.1: Rootstock M y c o r r h i z a l C o l o n i z a t i o n : A p r i l 1987 * same l e t t e r i n d i c a t e s no s i g n i f i c a n c e d i f f e r e n c e (a = 0.05 Mann-Whitney U t e s t ) 25 percent of a l l samples from this nursery were found to have 0% mycorrhizal colonization and 89% had less than 10% colonization in Fields 1, 2 and 3. Field 4 had 85% at less than 10 % colonization (Figure 2.9). The highest colonization rate for this nursery was 37% found in field 1 in a M 26 block. The nature of the experimental design was highly imbalanced, so statistical conclusions should be treated with caution. There were no difference in VAM colonization amongst the four fields, the blocks within each field and interactions amongst field, rootstock and blocks (Table 2.2a). There was however, a significant difference in colonization amongst the different rootstocks. M 2 differed significantly from a l l other rootstocks (Figure 2.10). The remaining rootstocks, showed no significant differences amongst each other. The pattern of mycorrhizal colonization were generally the same for Fields 1 and 2 (Figures 2.11 and 2.12). Colonization was low except for 2 to 3 isolated peaks, located along the perimeter of the field. Field 1 showed 3 peaks: 37% in the M 26c block, and 19% and 17% in the M 26a block (Figure 2.11). There was a minor peak of 8% in the center of the field in the MM 111 block. Field 2 also showed high peaks of 23% in the M 26b block and 19% in the M 26a block, both on the field's perimeter. Toward the center of the field a peak of 17% was found in the M 2 block (Figure 2.12). The distribution of mycorrhizae in Fields 3 and 4 were similar (Figures 2.13 and 2.14). Like Field 1 and 2, the highest 26 I # SAMPLES Field 1: 37 Field 2: 29 Field 3: 44 Field 4: 77 _CSL 1 - 10 11 - 20 21 - 30 31 - 40 > 40 PERCENT COLONIZATION Field 1 • Field 2 • Field 3 H Field 4 Figure 2.9: VAM D i s t r i b u t i o n i n Stoolbeds by F i e l d : F a l l 1987 O o o MM 111 M 4 M 9 Ottawa 3 MM 106 M 2 M 7 M26 Alnarp 2 ROOTSTOCK F i g u r e 2.10: Rootstock C o l o n i z a t i o n , Traas Nursery: F a l l 1987 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e s (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y F i e l d 3 222.360 74.120 0.923 0.460 Rootstock 8 1215.200 151.910 5.753 0.008 F i e l d * R s t o c k 9 237.660 26.407 0. 329 0.949 B l o c k ( F i e l d , 12 964.380 80.365 1.506 0.128 Roostock) E r r o r 149 7952.700 53.374 T o t a l 181 10468.000 Ta b l e 2.2: A n a l y s i s of V a r i a n c e f o r VAM i n Traas Nursery 28 3 7 Figure 2.11: Patterns of VA Mycorrhizae i n Stoolbeds: Traas Nursery F i e l d 1 29 47 Figure 2.13: Patterns of VA Mycorrhizae i n Stoolbeds: Traas Nursery F i e l d 3 31 p e r c e n t c o l o n i z a t i o n was g e n e r a l l y l o c a t e d a l o n g the p e r i p h e r y of the f i e l d s . In F i e l d 3, a peak o f 47% was found i n the M 2 b l o c k ( F i g u r e 2.13). In the M 7 b l o c k t h e r e were peaks o f 13% l o c a t e d a l o n g the p e r i p h e r y of the f i e l d . Both M 26 b l o c k s had h i g h e r than 10% c o l o n i z a t i o n ; b l o c k had 10% and 18% peaks found i n the c e n t e r of the f i e l d and b l o c k b had 10% and 23% c o l o n i z a t i o n . F i e l d 4 showed t h e most v a r i a t i o n i n m y c o r r h i z a l c o l o n i z a t i o n o f a l l 4 f i e l d s ( F i g u r e 2.14). There were 12 peaks of 10 % or g r e a t e r found i n t h i s f i e l d . The h i g h e s t peaks o f 25%, 28% and 32% were found along the pe r i m e t e r o f the f i e l d , i n b l o c k s M 7b, Ottawa 3a and MM 111b r e s p e c t i v e l y . BUDDED NURSERY M y c o r r h i z a l S t a t u s o f Apple Rootstocks While t h e r e was a s i g n i f i c a n t d i f f e r e n c e i n m y c o r r h i z a l p o p u l a t i o n between the two n u r s e r i e s , t h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n VAM c o l o n i z a t i o n o f r o o t s t o c k s w i t h i n the budded nursery ( F i g u r e 2.15). D i s t r i b u t i o n o f VAM Fungi T h e r e was a s i g n i f i c a n t d i f f e r e n c e i n m y c o r r h i z a l c o l o n i z a t i o n between the s t o o l b e d and budded n u r s e r y . In the budded nursery t h e r e were no samples c o l l e c t e d t h a t had 0% c o l o n i z a t i o n . The lowest percent c o l o n i z a t i o n found i n t h i s n u r s e r y was 30% w h i l e the h i g h e s t was 73%. There was no s i g n i f i c a n t d i f f e r e n c e i n mycorrhizae between the 2 f i e l d s , or amongst the 6 r o o t s t o c k s used i n t h i s study (Table 2.3a and 2.3b). 32 3 2 Figure 2.14: Patterns of VA Mycorrhizae i n Stoolbeds: Traas Nursery F i e l d 4 33 g 50 -O o UJ o M4 M 7 M 9 M 26 M 27 Alnarp 2 ROOTSTOCK F i g u r e 2.15: M y c o r r h i z a l C o l o n i z a t i o n i n Cannor Nursery * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y F i e l d 1 143.770 143.770 1.217 0.331 Rootstock 5 1611.100 322.210 2.727 0.176 B l o c k ( F i e l d , 4 472.530 118.130 1.406 0.242 Rootstock) E r r o r 65 5460.000 84.000 T o t a l 75 8665.400 Table 2.3: A n a l y s i s of V a r i a n c e f o r Mycorrhizae: Cannor Nursery 34 67 Figure 2.16: Patterns of VA Mycorrhizae: Cannor Nursery Field 1 35 The p a t t e r n of mycorrhizae i n F i e l d 1 d i d not f o l l o w t h a t of any f i e l d from the s t o o l b e d n u r s e r y . While the peak of 67% was found on the western perimeter of the f i e l d , t h e r e were other peaks of 55% or h i g h e r towards the e a s t e r n p a r t o f the f i e l d ( F i g u r e 2.16). Towards the c e n t r a l p a r t of the f i e l d , the c o l o n i z a t i o n r a t e dropped o f f though not below 30% ( F i g u r e 2.16). As i n o t h e r f i e l d s , the h i g h e s t m y c o r r h i z a l p o p u l a t i o n i n F i e l d 2 of the budded nursery was found along the p e r i m e t e r of the f i e l d . Peaks of 73% and 75% were found i n t h i s f i e l d along the n o r t h e a s t e r n and south western p a r t s of the f i e l d ( F i gure 2.17). C o l o n i z a t i o n d i d drop o f f towards the c e n t r a l p o r t i o n of the f i e l d though t h e r e appeared t o be a p l a t e a u of l e s s e r c o l o n i z a t i o n r a t e s on the e a s t e r n s i d e of the f i e l d . As i n F i e l d 1 the c o l o n i z a t i o n r a t e d i d not drop below 30%, the lowest percent c o l o n i z a t i o n i n the f i e l d was 36%. C o l d Storage and Rootstock Mycorrhizae Before c o l d storage M 4 and MM 111 had mycorrhizae present i n a l l 10 samples examined. M a i l i n g 26, M 7 and MM 106 had at l e a s t 2 samples wi t h 0% m y c o r r h i z a l c o l o n i z a t i o n . A f t e r 15 weeks of c o l d s t o r a g e , a l l r o o t s t o c k examined had a t l e a s t 1 sample w i t h 0% m y c o r r h i z a l c o l o n i z a t i o n . M a i l i n g 4 and MM 111 were the o n l y 2 r o o t s t o c k s of the 5 examined t h a t showed a s i g n i f i c a n t decrease i n p e r c e n t mycorrhizae due t o c o l d s t o r a g e (Table 2.4). DISCUSSION T h i s i s the f i r s t r e p o r t of d i f f e r e n t c l o n a l r o o t s t o c k s forming d i f f e r e n t percent m y c o r r h i z a l c o l o n i z a t i o n s i n apple 36 gure 2.17: Patterns of VA Mycorrhizae: Cannor Nursery F i e l d 2 Before After M 4 23 a* 9 b M 7 15 a 19 a M 26 22 a 23 a MM 106 13 a 11 a MM 111 18 a 8 b Table 2.4: VAM Colonization Before and After Cold Storage i n Rootstock from Stoolbeds * same l e t t e r indicates no s i g n i f i c a n t difference (a = 0.05 Mann-Whitney U test) 38 r o o t s t o c k from a s t o o l b e d n u r s e r y . T r a q u a i r and Berch (1988) and M i l l e r e t a l . (1985a) r e p o r t e d t h a t t h e r e was no d i f f e r e n c e i n VAM c o l o n i z a t i o n amongst d i f f e r e n t c l o n a l peach and apple r o o t s t o c k . The c l o n a l r o o t s t o c k t h a t these authors examined came from p l a n t s a l r e a d y e s t a b l i s h e d i n the orcha r d and not from nu r s e r y beds. In s t a b l e environments such as o r c h a r d s , the i n d i g e n o u s p o p u l a t i o n o f VAM f u n g i i s d i v e r s e and w e l l e s t a b l i s h e d . Under these s i t u a t i o n s , t h e r e may be no d i f f e r e n c e i n c l o n a l r e c e p t i v e n e s s t o VAM f u n g i . D i f f e r e n t c u l t i v a r s of c l o n a l s t r a w b e r r i e s i n the nursery a l s o showed no d i f f e r e n c e i n m y c o r r h i z a l c o l o n i z a t i o n (Robertson e t a l . 1988). However, the r o o t systems o f the v a r i o u s c l o n a l s t r a w b e r r i e s do not d i f f e r as much as the r o o t systems amongst c l o n a l a p p l e s . Granger et al. (1983) proposed t h a t MM 111 and M 7 may d i f f e r i n growth response t o VAM i n o c u l a t i o n , but they do not d i f f e r i n r e c e p t i v e n e s s t o VAM f u n g i . However, these t e s t s were done i n v i t r o . In f i e l d a n a l y s i s such as t h i s s t u d y , t h e r e may be a d i f f e r e n t r e c e p t i v e n e s s of the c l o n e s t o VAM f u n g i . Granger et a l . (1983) a t t r i b u t e the d i f f e r e n t growth responses t o the d i f f e r e n t r o o t systems o f these two apple c l o n e s . While the ro o t morphology o f each r o o t s t o c k was not examined i n t h i s study, i t i s p o s s i b l e t h a t the d i f f e r e n t r o o t systems found i n c l o n a l s t o c k may i n f l u e n c e the r e c e p t i v e n e s s of the host t o the fungus. There are many f a c t o r s t h a t i n f l u e n c e r o o t development o f apples i n the n u r s e r y . In s t o o l b e d s , h i g h h u m i d i t y , deepness o f h i l l e d s o i l and low l i g h t favours the p r o d u c t i o n o f r o o t s and burrknots (Rom and Brown 1979, Rom and Motichek 1987). One of the major f a c t o r s i n f l u e n c i n g r o o t development i s the p r o d u c t i o n of b u r r k n o t s . Burrknots are nonpathogenic c l u s t e r s of r o o t i n t i a l s c a p a b l e o f f o r m i n g r o o t s under f a v o u r a b l e c o n d i t i o n s . D i f f e r e n t apple c l o n e s produce d i f f e r e n t amounts of b u r r k n o t s , M 2 c o n s i s t e n t l y producing fewer than o t h e r r o o t s t o c k s (Rom and Brown 1973). D i f f e r e n t apple c l o n e s a l s o produce d i f f e r e n t l e v e l s of auxin and g i b b e r e l l i n , low v i g o u r c l o n e s showed h i g h e r l e v e l s of auxins and g i b e r e l l i n s than h i g h v i g o u r c l o n e s (Grochowska e t a l . 1984) . I n o c u l a t i o n of lemon and t a m a r i l l o w i t h VAM f u n g i induced r o o t i n i t i a t i o n and development i n the absence of r o o t i n g hormones (Cooper 1983), i n d i c a t i n g t h a t these f u n g i i n f l u e n c e l e v e l s of growth hormones i n r o o t s of t r e e s . M a i l i n g 2 c o n s i s t e n t l y showed h i g h e r VAM c o l o n i z a t i o n than the o t h e r r o o t s t o c k s . The h i g h i n c i d e n c e of the VAM fungus i n t h i s c l o n e may i n f l u e n c e the amounts of growth hormones produced t h e r e f o r e i n f l u e n c i n g the amounts of b u r r k n o t s and i t s r o o t development. M a i l i n g 2 spread over a l a r g e r s o i l area i n 5 d i f f e r e n t s o i l s than any o t h e r r o o t s t o c k (Coker 1958). I t i s t h e r e f o r e p o s s i b l e t h a t the VAM fungus i n f l u e n c e s the s i z e of the r o o t system of the c l o n e . The l a r g e r the r o o t system, the more s o i l area the c l o n e i s a b l e t o reach and t h e r e f o r e the p r o b a b i l i t y of encountering l i v e VAM propagules i s i n c r e a s e d . In b o t h n u r s e r i e s , t h e h i g h e s t p e r c e n t m y c o r r h i z a l 40 c o l o n i z a t i o n i s found along or towards the p e r i m e t e r of each f i e l d . T r a f f i c and t h e r e f o r e l i v e sources of inoculum, i s g r e a t e s t along the p e r i m e t e r s . I t would appear as i f the VAM f u n g i are moving inwards i n each f i e l d . In the budded n u r s e r y , the f i e l d s were never fumigated a l l o w i n g the indigenous VAM f u n g i t o become w e l l e s t a b l i s h e d . The f u n g i may be s e t back through the summer a f t e r an a p p l i c a t i o n of f u n g i c i d e , but i n d i c a t i o n s are t h a t the f u n g i are e i t h e r not a f f e c t e d , or i f they a r e , they are a b l e t o reinvade the s e e d l i n g s . I t has been hypothesized t h a t the a d d i t i o n s of c e r t a i n f u n g i c i d e s may i n c r e a s e r o o t exudates, thus i n c r e a s i n g the VAM c o l o n i z a t i o n (Menge 1982). The r o o t i n g depth of the budding nu r s e r y i s a l s o deeper than the s t o o l b e d n u r s e r y . The f u n g i i n the budding nursery may t h e r e f o r e be p h y s i c a l l y p r o t e c t e d from s o i l a p p l i c a t i o n s of nonsystemic f u n g i c i d e s and may even be s t i m u l a t e d by r e l e a s e s o f r o o t exudates a f t e r f u n g i c i d e a p p l i c a t i o n . Fumigation of the s o i l e l i m i n a t e s VAM f u n g i and t h i s i s p r o b a b l y the primary reason f o r the low c o l o n i z a t i o n r a t e i n the s t o o l b e d n u r s e r y . Two t o s i x months a f t e r f u m i g a t i o n , VAM f u n g i are a b l e t o r e e s t a b l i s h i n s o i l s (Menge 1982). Many of the s t o o l b e d s i n the nursery used i n t h i s study were o l d e r than 5 y e a r s . Mycorrhizae should have become e s t a b l i s h e d i n the f i e l d by t h i s t i m e . The s t o o l b e d s are sprayed f o r f u n g a l pathogens 5 - 7 times d u r i n g the season. I t i s most l i k e l y t h a t s p r a y i n g the s t o o l b e d s throughout the season may be a d v e r s e l y a f f e c t i n g the 41 VAM f u n g i . While t h e r e may be VAM f u n g i p r e s e n t i n the s t o o l b e d nursery they are never allowed t o p r o l i f e r a t e and spread throughout the f i e l d . Management p r a c t i c e s i n h a r v e s t i n g and p r o c e s s i n g of rootocks from s t o o l b e d s may a l s o be d e t r i m e n t a l to the VAM f u n g i . Nemec (1987) r e p o r t e d t h a t s t o r i n g VAM p l a n t s a t 5 - 10°C d i d not a f f e c t the a b i l i t y o f the f u n g i t o spread through c i t r u s r o o t s . The temperature under which r o o t s t o c k s used i n t h i s study were s t o r e d should not be a f f e c t i n g VAM c o l o n i z a t i o n . However, hi g h moisture d u r i n g prolonged storage appears t o be d e t r i m e n t a l to v i a b i l i t y o f VAM f u n g i as t h i s l e a d s t o d e t r i m e n t a l r e d u c t i o n s of oxygen t e n s i o n (Nemec 1987) . The humidity i n the c o l d storage rooms i s maintained a t over 75%. Such h i g h h u m i d i t i e s c o u l d be d e l e t e r i o u s t o VAM c o l o n i z a t i o n i n those r o o t s t o c k s t h a t are s t o r e d beyond a s p e c i f i c t h r e s h o l d time p e r i o d . M a i l i n g Merton 111 was the f i r s t r o o t s t o c k t o be p l a c e d i n c o l d s t o r a g e and was t h e r e f o r e under the most s t r e s s . Reduced 02 c o n c e n t r a t i o n i n t h i s p a r t of the c o l d room c o u l d have had d e t r i m e n t a l e f f e c t s on the VAM f u n g i . E i t h e r the temperature, the h u m i d i t y , 0 2 l e v e l , or d u r a t i o n of s t o r a g e may have reduced the VAM f u n g i i n t h i s r o o t s t o c k . M a i l i n g 4 was not p l a c e d i n c o l d s t o r a g e , but simply s t o r e d i n a c o o l dry shed. Reid and Bowen (1979) found t h a t under very dry s i t u a t i o n s , VAM c o l o n i z a t i o n dropped d r a s t i c a l l y . In t h i s i n s t a n c e the M 4 r o o t s may have been too dry d u r i n g s t o r a g e , c a u s i n g a r e d u c t i o n i n r o o t c o l o n i z a t i o n . 42 The m y c o r r h i z a l s t a t u s of apple r o o t s t o c k c l o n e s i n and l e a v i n g s t o o l b e d n u r s e r i e s v a r i e s . However, the v a r i a t i o n i n mycorrhizae found i n t h i s study i s more l i k e l y t o be a r e s u l t of management p r a c t i c e s and p o s i t i o n of the r o o t s t o c k i n the f i e l d i n r e l a t i o n t o sources of inoculum than t o a c t u a l genotype. In areas where t h e r e i s h i g h inoculum such as a l o n g the p e r i m e t e r of the f i e l d s , the r o o t s are more l i k e l y t o come i n c o n t a c t w i t h the inoculum. As r o o t s t o c k s are p l a n t e d by b l o c k , r a t h e r than randomly the m y c o r r h i z a l c o l o n i z a t i o n of each c l o n e depends on i t s p o s i t i o n i n r e l a t i o n t o inoculum s o u r c e . Management p r a c t i c e s i n t h i s s t o o l b e d nursery i n c l u d i n g high r a t e s of f e r t i l i z a t i o n , use of p e s t i c i d e s , h a r v e s t i n g time and s t o r a g e may be d e t r i m e n t a l t o VAM i n c e r t a i n c l o n a l r o o t s t o c k s . The environment i n t h i s s t o o l b e d nursery does not favour indigenous VAM f u n g i , w h i l e i n budded n u r s e r i e s i t does. I t may be necessary t o a l t e r management p r a c t i c e s , s p e c i f i c a l l y t o reduce the amounts of p e s t i c i d e s used, t o ensure t h a t VAM f u n g i a r e n o t d e t r i m e n t a l l y a f f e c t e d . A l t e r n a t i v e l y , i n o c u l a t i n g s t o o l b e d s w i t h VAM f u n g i w i l l a i d i n the p r o p a g a t i o n of VAM f u n g i and thereby i n c r e a s i n g the p r o b a b i l i t y of the c l o n e s becoming m y c o r r h i z a l . I f VAM f u n g i do p l a y a r o l e i n the e s t a b l i s h m e n t of h e a l t h y o r c h a r d s , r o o t s t o c k t h a t spends time i n the budding nursery should perform b e t t e r i n the o r c h a r d than r o o t s t o c k d i r e c t l y from s t o o l b e d n u r s e r i e s . I f o r c h a r d s o i l s are fumigated, VAM i n o c u l a t i o n s i n the nursery should be c a r r i e d o u t . 43 STERILIZING OLD APPLE SOILS INCREASES PLANT HEIGHT IN POT TESTS: CONFIRMATION OF THE PRESENCE OF APPLE REPLANT DISEASE INTRODUCTION Crops p l a n t e d i n t o s o i l t h a t p r e v i o u s l y supported the same crop w i l l o f t e n show a 1 s i c k n e s s1 t h a t i s e a s i l y overcome by s t e r i l i z i n g the s o i l (Deal e t a l . 1971, Hwang 1988, Pepin e t a l . 1975, T r u d g i l l 1984). T h i s d i s o r d e r i s e s p e c i a l l y common i n o l d apple s o i l s (Covey e t a l . 1984, Hoestra 1968, Ross e t a l . 1984, Savory 1967, S l y k h u i s and L i 1985). In the absence o f an i d e n t i f i a b l e pathogen, t h i s s i c k n e s s i n o l d apple s o i l s has been termed 'Apple Replant D i s e a s e ' (ARD). The i n c r e a s e i n shoot h e i g h t due t o s t e r i l i z a t i o n i n d i c a t e s t h a t a b i o l o g i c a l agent may be i n v o l v e d w i t h t h i s problem. S l y k h u i s and L i ' s study (1985), i n d i c a t i n g t h a t phosphorus f e r t i l i z e r s may a l s o a l l e v i a t e t h i s problem suggests t h a t v e s i c u l a r - a r b u s c u l a r m y c o r r h i z a l (VAM) f u n g i may p l a y a r o l e i n ARD. To e l i m i n a t e the s p e c u l a t i o n on when ARD appears, pot bi o a s s a y s are be i n g used t o p r e d i c t i f a s o i l has ARD. Hoestra (1968) developed the b a s i c b i o a s s a y arid v a r i a t i o n s are used u n i v e r s a l l y (Ryan 1975, Sewell and White 1981, S l y k h u i s and L i 1985, Upstone 1974) . H a l f of the t e s t s o i l i s fumigated. Apple s e e d l i n g s , i n the 1 - 2 l e a f stage are t r a n s p l a n t e d i n t o both fumigated and nonfumigated s o i l . I f t h e r e i s a s i g n i f i c a n t i n c r e a s e i n shoot h e i g h t i n the fumigated p o t s compared t o the nonfumigated, i n the absence o f an i d e n t i f i a b l e pathogen, the s o i l i s i d e n t i f i e d as being ARD. Very s p e c i f i c s o i l h a n d l i n g and t e s t i n g methods are used i n pot b i o a s s a y s . For i n s t a n c e , s o i l s s h ould be c o l l e c t e d from under the o r c h a r d canopy, p r e f e r a b l y i n the f a l l and must be kept c o o l and moist u n t i l use. S o i l from one o r c h a r d i n t h i s study was a i r d r i e d , t o determine i f a i r - d r y i n g would i n t e r f e r e w i t h the outcome of ARD pot b i o a s s a y s . S l y k h u i s and L i (1985) found t h a t i n c r e a s e s i n t e s t shoot h e i g h t o c c u r r e d a f t e r a number of b i o c i d e s and combinations of b i o c i d e s and f e r t i l i z e r s were used. While the change i n shoot h e i g h t due t o s o i l treatment i s w e l l documented, l i t t l e i s known of how these treatments a f f e c t s o i l n u t r i e n t s , VAM c o l o n i z a t i o n of s e e d l i n g r o o t s , and r o o t dry weight. I f s t e r i l i z a t i o n i s t o be used i n b i o a s s a y s , i t i s important t o understand i t s e f f e c t s on s o i l pH and n u t r i e n t a v a i l a b i l i t y . The e f f e c t s o f s t e r i l i z a t i o n on s o i l n u t r i e n t s v a r i e s depending on the s o i l and the type of s t e r i l a n t used. S o i l s h i g h i n o r g a n i c matter w i l l have a f l u s h of n i t r o g e n (N) and phosphorus (P) a f t e r s t e r i l i z a t i o n (Eno and Popenoe 1963, Skipper and Westermann 1973, Warcup 1957). A u t o c l a v i n g and steam s t e r i l i z i n g i n c r e a s e s o i l P more than o t h e r methods of s t e r i l i z a t i o n , but decrease the pH (Skipper and Westermann 1973). In o t h e r s o i l s , s t e r i l i z a t i o n by a u t o c l a v i n g does not a f f e c t s o i l pH or P but o n l y Mn (Gennari et a l . 1987, W i l l i a m s - L i n e r a and Ewel 1984). A i r - d r y i n g the s o i l s f o r extended p e r i o d s i n c r e a s e s the r a t e of o r g a n i c matter decomposition upon r e w e t t i n g and the subsequent r e l e a s e of s o i l n u t r i e n t s e s p e c i a l l y N and P ( L i e g e l 1983, Mack 1963). However, s t e r i l i z a t i o n e f f e c t s on N and pH are more pronounced i n moist s o i l s than i n dry s o i l s ( S a l o n i u s e t a l . 1967). T h i s study was t h e r e f o r e undertaken t o c o n f i r m S l y k h u i s and L i ' s (1985) f i n d i n g s t h a t s o i l s t e r i l z i a t i o n and added phosphorus w i l l i n c r e a s e p l a n t h e i g h t i n o l d apple s o i l s ; t o determine how s t e r i l i z a t i o n a f f e c t s s o i l pH and a v a i l a b l e P; t o determine how a i r - d r y i n g a f f e c t s s e e d l i n g growth i n pot b i o a s s a y s ; and to determine the e f f e c t s o f s o i l treatments on VAM c o l o n i z a t i o n o f s e e d l i n g r o o t s . MATERIAL AND METHODS COLLECTING SOILS FROM ORCHARDS S o i l s 1 - 4 S o i l s 1 - 4 were i d e n t i f i e d as having ARD a f t e r b e i n g t e s t e d by Dr. J . S l y k h u i s a t h i s Apple Replant T e s t i n g S e r v i c e i n Summerland, B.C. During the f i r s t week i n May 1987, the s o i l s were c o l l e c t e d from the f o l l o w i n g orchards: S o i l 1, from C l a r k B r o t h e r s , Upper Bench Rd., RR# 1, Keremeos, B.C.; S o i l 2 from Bahnson B r o t h e r s , Black Sage Rd., RR# 1, O l i v e r B.C.; S o i l 3 from H a r l e q u i n Farms, Naramata Rd. , Naramata B.C.; S o i l 4 from Lammers Orchard, 10th Ave., RR# 1, Keremeos, B.C. (Table 3.1). At the time o f s o i l c o l l e c t i o n , o l d t r e e s had been removed from a l l o r c h a r d s . S o i l 1 had been r e p l a n t e d w i t h M 26 apple r o o t s t o c k . The new rows o f t r e e s had been p l a n t e d d i r e c t l y i n t o the o l d rows. I t was t h e r e f o r e easy t o i d e n t i f y the rows and alleyways from the p r e v i o u s c r o p . S o i l s 2 and 4 had been L o n g i t i . L a t i t u d e S o i l S e r i e s S o i l D e s c r i p t i o n S o i l 1 4 9 ° 13' 1 1 9 ° 49' Rutland G r a v e l l y Sandy Loam Sur f a c e s o i l s are dark brown wit h v a r i n g amounts of stones i n the low-p a r t s . U n d e r l y i n g m a t e r i a l s s t r a t i f -i e d sand & g r a v e l . S o i l 2 4 9 ° 9' 1 1 9 ° 32' Osoyoos Loamy Sand Su r f a c e s o i l s are brown; p r o g r e s s i v e r e d u c t i o n of s i l t & c l a y content t o bottom of solum. Lime accumulates i n lower B h o r i z o n S o i l 3 4 9 ° 34' 1 1 9 ° 30* P e n t i c t o n S i l t y Loam Solum t o depth of of about 100 cm i s g r e y s i s h s i l t loam Substratum compos-ed of deep beds of s t r a t i f i e d s i l t y , c l a y & f i n e sand S o i l 4 4 9 ° 12' 1 1 9 ° 50' N i s c o n l i t h Loam There i s a d e c i d -uous l e a f l a y e r a t s o i l s u r f a c e . Deep A h o r i z o n , f o l l o w -ed by a s t r u c t u r e -l e s s G h o r i z o n . S o i l 5 4 9 ° 57' 1 1 9 ° 22' Rutland G r a v e l l y Sand Loam Sur f a c e s o i l dark brown shading t o brown i n lower l a y e r , w i t h v a r y -i n g amounts of stones and g r a v e l . T a b l e 3.1: S i t e L o c a t i o n and D e s c r i p t i o n of Orchards ( K e l l y and S p i l s b u r y 1949) 47 ploughed, d i s c e d and summer f a l l o w e d . S o i l 2 had a dense cover of b r o a d l e a f and g r a s s weeds, w h i l e S o i l 4 had been seeded w i t h b a r l e y . At s i t e 3, the s o i l had been ploughed and d i s c e d the p r e v i o u s day so t h e r e was no cover c r o p . An attempt was made t o keep the s o i l s c o l l e c t e d from o l d rows and a l l e y w a y s s e p a r a t e , i n c a s e t h e pH d i f f e r e d s i g n i f i c a n t l y between these two areas i n the o r c h a r d . Only at s i t e 1 was t h i s p o s s i b l e . For the remaining o r c h a r d s , o l d rows had t o be estimated by a l i g n i n g rows from adjacent b l o c k s . S o i l was c o l l e c t e d from f i f t e e n s i t e s from the a l l e g e d rows and 15 s i t e s from the a l l e g e d a l l e y w a y s , f o r a t o t a l of 30 s i t e s per o r c h a r d . At each c o l l e c t i o n s i t e p o l y e t h y l e n e bags were f i l l e d with approximately 1 l i t r e of s o i l from the top 15 cm. A l l p l a n t s and l a r g e r o o t s were removed. A l l bags were l a b e l l e d and brought t o the l a b o r a t o r y a t the U n i v e r s i t y of B r i t i s h Columbia (UBC) where they were s t o r e d i n a c o o l dry a r e a . A subsample o f about 500 gm of s o i l was taken from each bag, a i r - d r i e d , and the pH determined. S i n c e no d i f f e r e n c e i n s o i l pH f o r the rows and alleyways was e v i d e n t (Appendix), a composite sample f o r each o r c h a r d was made. The s o i l from each bag was passed through a 2 cm s i e v e t o remove the l a r g e r p i e c e s o f d e b r i s . A l l composite samples were then a i r - d r i e d a t 22°C over a p e r i o d of 7 days. Once a l l s o i l s were a i r - d r i e d they were s t o r e d i n l a r g e c o n t a i n e r s u n t i l f u r t h e r use. S o i l 5 4 8 S o i l 5 had been i d e n t i f i e d as having ARD by pot and f i e l d t e s t s done by Dr. G. N e i l s e n o f A g r i c u l t u r e Canada, Summerland B.C. and Dr. J . Yorston o f B.C. M i n i s t r y o f A g r i c u l t u r e and F i s h e r i e s , Kelowna, B.C. During the t h i r d week of September 1987, S o i l 5 was c o l l e c t e d from Ummard's o r c h a r d , S p r i n g e r Ave., Kelowna, B.C. (Table 3.1). The s o i l was c o l l e c t e d from a row of t r e e s t h a t had been used i n ARD f i e l d t r i a l s . Not a l l of the o l d t r e e s from the row had been removed. S o i l was c o l l e c t e d from under the canopy, about 1 - 1.5 meters from the trunk o f the o l d t r e e a v o i d i n g t r e a t e d a r e a s . The top 7 - 8 cm of s o i l i n c l u d i n g the gras s cover crop was d i s c a r d e d . S o i l t o a depth of 30 cm, i n c l u d i n g r o o t s from the apple t r e e , was p l a c e d i n 20 l i t r e c o n t a i n e r s . A l l f i l l e d c o n t a i n e r s were brought back t o the l a b o r a t o r y at UBC. A composite sample was made from a l l s o i l c o l l e c t e d from t h i s o r c h a r d . S o i l was passed through a 2 cm s i e v e . H a l f o f the composite sample was a i r - d r i e d at 22°C over a p e r i o d o f 7 days, and then s t o r e d . The othe r h a l f was kept moist i n a c o o l dark c o n t a i n e r u n t i l f u r t h e r use. POT BIOASSAY TESTS FOR ARD T e s t S e e d l i n g s O p e n - p o l l i n a t e d Red D e l i c i o u s or Macintosh apple seeds t r e a t e d w i t h Captan were used i n a l l pot t e s t s . Seeds were ob t a i n e d from the v i r u s - f r e e orchard of A g r i c u l t u r e Canada Research S t a t i o n i n Summerland, B.C. The seeds were wrapped i n moist paper t o w e l s , p l a c e d i n p o l y e t h y l e n e bags and v e r n a l i z e d i n the r e f r i g e r a t o r f o r 10 - 14 weeks. Once r a d i c l e s had emerged from 80% o f the seeds, they were p l a n t e d i n t r a y s c o n t a i n i n g moist c a l c i n e d m o n t m o r i l l o n i t e c l a y ( T u r f a c e , A p p l i e d I n d u s t r i a l M a t e r i a l s Corp., D e e r f i e l d I I . ) . The s e e d l i n g s grew f o r about 1 week t o the 1 -2 l e a f stage and were then used f o r pot experiments. S o i l s 1 - 4 A l l s o i l s were t r e a t e d i d e n t i c a l l y . One h a l f o f each composite sample was s t e r i l i z e d , and t o both the s t e r i l i z e d and non s t e r i l i z e d s o i l , 3 f e r t i l i z e r treatments were added. The f e r t i l i z e r s used were monoammonium phosphate (11-55-0), ammonium n i t r a t e (34-0-0) and t r i p l e superphosphate (0-45-0). Four a u t o c l a v a b l e and 4 l a r g e p o l y e t h y l e n e bags were each f i l l e d w i t h 4 l i t r e of a i r - d r i e d s o i l from each o r c h a r d . Enough water (500 ml) was added t o each bag t o moisten the s o i l . The 4 a u t o c l a v a b l e bags were a u t o c l a v e d f o r 1 hour a t 94 kPa a t 12 0°C. The bags were kept at 22°C f o r 24 hours and then a u t o c l a v e d again as above. They were removed from the a u t o c l a v e , opened and allowed t o a e r a t e f o r 3 days a l l o w i n g any v o l a t i l e p h y t o t o x i c substances t o escape. The p o l y e t h y l e n e bags were s t o r e d u n t i l pot t e s t s were begun. Three hundred mis of s t e r i l i z e d o r n o n s t e r i l i z e d s o i l were p l a c e d i n 8 cm p o t s . One apple s e e d l i n g was t r a n s p l a n t e d i n t o each p o t . There were 6 r e p l i c a t e s per treatment. The v a r i o u s f e r t i l i z e r s a t r a t e s used i n standard ARD pot b i o a s s a y s were 50 Figure 3.1: P l a n t Growth Bench added t o the t e s t pots: 11-55-0 at 1.5 gm/1 (0.165 gm N and 0.3 6 gm P) , 34-0-0 at 0.45 gm/1 (0.15 gm N) and 0-45-0 at 1.8 gm/1 (0.35 gm P) (Slykhuis and L i 1985). The f e r t i l i z e r s were incorporated i n t o the s o i l without d i s t u r b i n g the s e e d l i n g . Each pot was watered thoroughly and placed on a p l a n t growth bench (Figure 3.1). The seedlings were grown under 215 rimol/m 2/sec l i g h t at 25°C w i t h 16 hour d a y l i g h t . During the f i r s t 2 weeks a f t e r t r a n s p l a n t i n g , s e e d l i n g s that had d i e d were replaced. A f t e r 2 weeks, dead s e e d l i n g s were considered t o be a r e s u l t of treatment. Ten weeks a f t e r t r a n s p l a n t i n g , shoot height was measured f o r each treatment. Roots from S o i l s 1 and 2 were f i x e d i n FAA f o r myc o r r h i z a l counts and root dry weights. A f t e r m y c o r r h i z a l counts were made, root fragments f o r each sample were placed on l a b e l l e d f i l t e r 51 papers, washed thoroughly with tap water and oven-dried a t 60°C f o r 5 days. A n a l y s i s of V a r i a n c e (ANOVA) was done f o r a l l t e s t s o i l s . S o i l 5 S o i l from t h i s o r c h a r d was used t o determine i f a i r - d r y i n g the s o i l may a f f e c t pot t e s t r e s u l t s . Ten a u t o c l a v a b l e bags were each f i l l e d w i t h 4 l i t r e s o f a i r - d r i e d (AD) and n o n a i r - d r i e d (NAD) s o i l . Both the AD and NAD s o i l s were g i v e n the f o l l o w i n g 5 treatments: 1) F o r m a l i n (FM) a t 25 mis 14.8% f o r m a l i n / 1 s o i l ( v o l / v o l ) ; ( S l y k h u i s and L i 1985); 2) A u t o c l a v i n g twice (Al) at 94 KPa a t 12 0°C f o r 1 hour each 24 hours a p a r t ; 3) A u t o c l a v i n g (A2) at 94 KPa at 120°C f o r 3 hours (Buszard and Jensen 1986); 4) P a s t e u r i z i n g (PA) a t 70°C f o r 1 hour i n a hot water bath ( S l y k h u i s and L i 1985) ; and 5) Untreated c o n t r o l (CT). Bags t r e a t e d w i t h f o r m a l i n were taped shut and incubated f o r 14 days. The bags were aerated f o r 21 days b e f o r e use i n pot t e s t s . With both a u t o c l a v e d treatments the bags were allowed t o a e r a t e f o r 3 days. One 8 cm pot r e p l i c a t e d 8 times was f i l l e d w i t h 300 mis of t r e a t e d or non-treated s o i l . One 1 - 2 l e a f stage apple s e e d l i n g was t r a n s p l a n t e d i n t o each p o t . The pots were thoro u g h l y watered and p l a c e d on the p l a n t bench. A f t e r 6 weeks, the h e i g h t of each s e e d l i n g was measured, and the r o o t s were f i x e d i n FAA. Percent m y c o r r h i z a l c o l o n i z a t i o n and r o o t dry weights were measured. A n a l y s i s of V a r i a n c e was conducted f o r a l l t r e a t m e n t s . VAM FUNGI 52 Examination of r o o t s f o r mycorrhizae was done as p r e v i o u s l y d e s c r i b e d (see Chapter 2 page 23) . SOIL ANALYSIS S o i l pH S o i l pH i n water (1:1) ( v o l : v o l ) was taken f o r a l l samples from orchards 1 - 4 p r i o r t o making composite samples. A f t e r s o i l s t e r i l i z a t i o n the pH was taken f o r each of the 5 orc h a r d s o i l s ( L a v k u l i c h 1978 pp 1) . For pH from rows and alleyways a F i s h e r Accumet Model 42 0 D i g i t a l pH/ion meter was used. For the pH from s t e r i l e and n o n s t e r i l e s o i l an O r i o n Research, Analog pH metre, Model 3 01 was used. The e l e c t r o d e was p l a c e d i n the supernatant and the r e a d i n g was allowed t o s t a b i l i z e b e f o r e r e c o r d i n g . S o i l Phosphorus The a v a i l a b l e P of each t r e a t e d and nontreated o r c h a r d s o i l used f o r pot experiments was analysed u s i n g the Olsen method as the p r e l i m i n a r y pH readings f o r these s o i l s were h i g h ( L a v k u l i c h 1978 pp 55-57) . RESULTS BIOASSAY TEST S e e d l i n g M o r t a l i t y S o i l 5 was the only s o i l i n which t h e r e was 100% s e e d l i n g s u r v i v a l . In S o i l s 1 - 4 t h e r e was some m o r t a l i t y of t e s t s e e d l i n g s , though t h e r e appeared t o be no p a t t e r n i n death of the s e e d l i n g due t o treatment. S o i l s 1 - 4 Shoot Height 53 There were s i g n i f i c a n t i n c r e a s e s i n p l a n t h e i g h t due to s t e r i l i z a t i o n f o r S o i l s 1 - 4 (Tables 3.2, 3.3, 3.4, and 3.5). A s i g n i f i c a n t i n t e r a c t i o n between s t e r i l i z a t i o n and f e r t i l i z e r o c c u r r e d o n l y i n S o i l 3 (Table 3.4). S t e r i l i z i n g t h i s s o i l r e s u l t e d i n an a d d i t i v e shoot i n c r e a s e o n l y i n the 0-45-0 treatment ( F i g u r e 3.4) In S o i l s 1, 2 and 3, f e r t i l i z e r s caused s i g n i f i c a n t i n c r e a s e s i n shoot h e i g h t ( F i g u r e s 3.2, 3.3 and 3.4). The f e r t i l i z e r s 11-55-0 and 34-0-0 caused s i g n i f i c a n t i n c r e a s e s i n shoot h e i g h t i n these 3 s o i l s w h i l e 0-45-0 d i d not ( F i g u r e s 3.2, 3.3 and 3.4). F e r t i l i z e r s d i d not cause any s i g n i f i c a n t changes i n shoot h e i g h t i n S o i l 4 (Table 3.5). There were i n c r e a s e s i n shoot growth i n the 0-45-0 treatment f o r S o i l s 1 and 3 o n l y a f t e r s t e r i l i z a t i o n . Root Dry Weight S t e r i l i z i n g S o i l 1 d i d not change r o o t dry weight, but adding 0-45-0 i n c r e a s e d r o o t dry weight s i g n i f i c a n t l y ( F igure 3.6) . Root dry weight d i d not change s i g n i f i c a n t l y due to s t e r i l i z a t i o n or the a d d i t i o n of f e r t i l i z e r i n S o i l 2 (Table 3.7) . There was no s i g n i f i c a n t i n t e r a c t i o n between s o i l s t e r i l i z a t i o n and f e r t i l i z e r f o r r o o t dry weight f o r e i t h e r S o i l 1 or 2 (Tables 3.6 and 3.7). VAM Fungi S t e r i l i z i n g the s o i l e l i m i n a t e d the VAM f u n g i i n both S o i l s 1 and 2 ( F i g u r e 3.8 and 3.9). In the n o n s t e r i l e treatments, 11-55-0 decreased the mycorrhizae s i g n i f i c a n t l y i n both s o i l s 54 30 -I Control Non Sterile 1 1 - 5 5 - 0 3 4 - 0 - 0 FERTILIZER 0 - 4 5 - 0 • Sterile F i g u r e 3.2: Mean Shoot Height a t 10 weeks: S o i l 1 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e f o r mean of each f e r t i l i z e r treatment (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean square F-• r a t i o P r o b a b i l i t y S t e r i l i z . 1 495. 49 495. 49 34. 89 0.000 F e r t i l i z e r 3 521. 15 173 . 72 12. 23 0.000 S t e r i l i z . * 3 34. 38 11. 46 0. 81 0.500 F e r t i l i z e r E r r o r 31 440. 30 14. 20 T o t a l 38 1448 . 00 Tabl e 3.2: A n a l y s i s o f V a r i a n c e f o r Shoot h e i g h t : S o i l 1 55 30 -, ! 25 : Control 1 1-55-0 34 -0 -0 0-45-0 FERTILIZER • Non Sterile • Sterile F i g u r e 3.3: Mean Shoot Height a t 10 weeks: S o i l 2 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e f o r mean of each f e r t i l i z e r treatment (a = 0.05 Tukey's (HSD) t e s t ) Sum of Mean F - r a t i o P r o b a b i l i t y Source DF squares square S t e r i l i z . 1 300.55 300.55 13.58 0.000 F e r t i l i z e r 3 438.65 146.22 6. 61 0. 000 S t e r i l i z . * 3 23 . 37 7.79 0.35 0.788 F e r t i l i z e r E r r o r 39 863.30 22.14 T o t a l 46 1641.20 Tabl e 3.3: A n a l y s i s of V a r i a n c e f o r Shoot Height: S o i l 2 56 Control 11-55-0 34-0 -0 0-45-0 FERTILIZER • Non Sterile • Sterile F i g u r e 3.4: Mean Shoot Height a t 10 weeks: S o i l 3 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y S t e r i l i z . 1 163.43 163.43 4.22 0.047 F e r t i l i z e r 3 1267.60 422.53 10.92 0. 000 S t e r i l i z . * 3 375.42 125.14 3.23 0. 033 F e r t i l i z e r E r r o r 37 1432.30 38.71 T o t a l 44 3310.80 Table 3.4: A n a l y s i s o f V a r i a n c e f o r Shoot Height: S o i l 3 57 a* a a a 30 -Control 1 1-55-0 34-0-0 0-45-0 FERTILIZER • Non Sterile • Sterile F i g u r e 3.5: Mean Shoot Height a t 10 weeks: S o i l 4 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e f o r mean of each f e r t i l i z e r treatment (a = 0.05 Tukey's (HSD) t e s t ) Sum of Mean F - r a t i o P r o b a b i l i t y Source DF squares square S t e r i l i z . 1 1070.50 1070.50 11. 52 0. 002 F e r t i l i z e r 3 531.27 177.09 1.91 0.145 S t e r i l i z . * 3 121.62 40. 54 0. 44 0. 728 F e r t i l i z e r E r r o r 38 3530.10 92.90 T o t a l 45 5293.20 Table 3.5: A n a l y s i s of V a r i a n c e f o r Shoot Height: S o i l 4 58 0.50 b Control 11-55-0 34-0 -0 0-45-0 FERTILIZER • Hon Sterile • Sterile F i g u r e 3.6: Mean Root Dry Weight a t 10 weeks: S o i l 1 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e f o r mean of each f e r t i l i z e r treatment (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y S t e r i l i z . 1 0.0029 0.0029 0.1226 0.729 F e r t i l i z e r 3 0.2244 0.0748 3.1584 0. 039 S t e r i l i z . * 3 0.0439 0.0146 0.6184 0. 608 F e r t i l i z e r E r r o r 31 0.7342 0.0236 T o t a l 38 1.0048 Table 3.6: A n a l y s i s of V a r i a n c e f o r Root Dry Weight: S o i l 1 59 0.50 i i l Control 11-55-0 34-0-0 0-45-0 FERTILIZER • Non Sterile • Sterile F i g u r e 3.7: Mean Root Dry Weight a t 10 weeks: S o i l 2 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e f o r mean of f e r t i l i z e r treatment (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y S t e r i l i z . 1 0.0196 0.0197 0.6050 0.441 F e r t i l i z e r 3 0.1415 0.0472 1.4494 0.243 S t e r i l i z . * 3 0.1166 0.0388 1.1940 0.325 F e r t i l i z e r E r r o r 39 1.2689 0.0324 T o t a l 46 1.5479 Table 3.7: A n a l y s i s o f V a r i a n c e f o r Root Dry Weight: S o i l 2 60 50 -, Control 11-55-0 34-0 -0 0-45-0 FERTILIZER • Hon Sterile • Sterile F i g u r e 3.8: Percent M y c o r r h i z a l C o l o n i z a t i o n : S o i l 1 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y S t e r i l i z . 1 8535.40 8535.30 88 .33 0. 000 F e r t i l i z e r 3 2154.50 718.20 7.43 0.001 S t e r i l i z . * 3 1824.80 608.25 6.30 0. 002 F e r t i l i z e r E r r o r 31 2995.70 96.64 T o t a l 38 15271.0 Ta b l e 3.8: A n a l y s i s of V a r i a n c e f o r VAM C o l o n i z a t i o n : S o i l 1 (Figure 3.8 and 3.9). The mycorrhizae i n the 34-0-0 and 0-45-0 treatments did not d i f f e r s i g n i f i c a n t l y from each other i n either S o i l s 1 or 2, but were s i g n i f i c a n t l y reduced from the controls i n S o i l 2 only (Figure 3.9). S o i l Analysis No s i g n i f i c a n t differences were found i n s o i l pH and s o i l P due to s t e r i l i z a t i o n i n S o i l s 1, 2, and 4 (Table 3.10c, 3.11c and 3.13c). There was a s i g n i f i c a n t decrease i n s o i l pH but not s o i l P a f t e r s t e r i l i z a t i o n i n S o i l 3 (Table 3.12c). AIR—DRYING/ARD POT BIOASSAY S o i l 5 Shoot Height A i r drying alone did not cause any s i g n i f i c a n t changes i n plant height (Table 3.14). However, pasteurizing and adding f o r m a l i n to the a i r - d r i e d s o i l i n c r e a s e d p l a n t height s i g n i f i c a n t l y (Figure 3.10). Neither of the autoclaving treatments increased plant height s i g n i f i c a n t l y . A s i g n i f i c a n t i n t e r a c t i o n between air-drying and s o i l s t e r i l i z a t i o n occurred (Table 3.14). Air-drying the s o i l increased the pasteurization and formalin e f f e c t s (Figure 3.10). However, ai r - d r y i n g the s o i l negated any increases i n plant height that may have occurred due to either autoclaving treatment (Figure 3.10). Root Dry Weight Air-drying the s o i l did not r e s u l t i n any s i g n i f i c a n t changes i n root dry weight (Table 3.15). However, pasteurizing and adding formalin to the s o i l s i g n i f i c a n t l y increased root dry Control 1 1-55-0 34-0 -0 0-45-0 FERTILIZER • Non Sterile • Sterile F i g u r e 3.9: Percent M y c o r r h i z a l C o l o n i z a t i o n : S o i l 2 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y S t e r i l i z . 1 10945.00 10945.00 319.14 0.000 F e r t i l i z e r 3 4460.40 1486.80 43 . 35 0. 000 S t e r i l i z . 3 4202.80 1400.90 40. 85 0. 000 F e r t i l i z e r E r r o r 39 1337.60 34.30 T o t a l 46 21324.00 Table 3.9: A n a l y s i s o f V a r i a n c e f o r VAM C o l o n i z a t i o n : S o i l 2 63 Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y S t e r i l i z . E r r o r T o t a l 1 4 5 0.027 0.467 0.494 0.027 0.117 0.229 0.658 Ta b l e 3.10a: A n a l y s i s o f V a r i a n c e f o r S o i l pH: S o i l 1 Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y S t e r i l i z . E r r o r T o t a l 1 4 5 31.510 663.542 695.052 31.510 165.885 0.190 0. 685 Ta b l e 3.10b: A n a l y s i s o f V a r i a n c e f o r S o i l P: S o i l 1 Non S t e r i l e S t e r i l e pH P (ppm) 6 . 1 a * 100.0 a 6.2 a 95.0 a * same l e t t e r w i t h i n i n category i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e s (a = 0.05 Tukey's (HSD) (t e s t ) T a b l e 3.10c: S o i l pH and P (Olsen Method) b e f o r e and a f t e r S t e r i l i z a t i o n : S o i l 1 64 Sum of Mean F - r a t i o P r o b a b i l i t y Source DF squares square S t e r i l i z . 1 0.027 0.027 0.165 0.705 E r r o r 4 0.647 0.162 T o t a l 5 0.674 Ta b l e 3 .11a: A n a l y s i s of V a r i a n c e f o r S o i l pH: S o i l 2 Sum of Mean F - r a t i o P r o b a b i l i t y Source DF squares square S t e r i l i z . 1 3.662 3.662 0.017 0.902 E r r o r 4 851.107 212.777 T o t a l 5 854.769 Table 3.11b: A n a l y s i s o f V a r i a n c e f o r S o i l P: S o i l 2 Non S t e r i l e S t e r i l e pH P (ppm) 5.7 a* 43.0 a 5.6 a 42.0 a * same l e t t e r w i t h i n i n category i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e s (a = 0.05 Tukey's (HSD) t e s t ) T a b l e 3.11c: S o i l pH and P (Olsen Method) b e f o r e and a f t e r S t e r i l i z a t i o n : S o i l 2 65 Sum of Mean F - r a t i o P r o b a b i l i t y Source DF squares square S t e r i l i z . 1 0.167 0.167 25.000 0. 007 E r r o r 4 0.027 0.007 T o t a l 5 0.194 Ta b l e 3. 12a: A n a l y s i s o f V a r i a n c e f o r S o i l pH: S o i l 3 Sum of Mean F - r a t i o P r o b a b i l i t y Source DF squares square S t e r i l i z . 1 84 . 375 84.375 0. 301 0.612 E r r o r 4 1120.833 280.208 T o t a l 5 1205.208 Table 3.12b: A n a l y s i s o f V a r i a n c e f o r S o i l P: S o i l 3 Non S t e r i l e S t e r i l e pH P (ppm) 6.2 a* 87.0 a 5.9 b 95.0 a * same l e t t e r w i t h i n i n category i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e s (a = 0.05 Tukey's (HSD) t e s t ) T a b l e 3.12c: S o i l pH and P (Olsen Method) b e f o r e and a f t e r S t e r i l i z a t i o n : S o i l 3 66 Sum of Mean F - r a t i o P r o b a b i l i t y Source DF squares square S t e r i l i z . 1 0.082 0.082 1.140 0.346 E r r o r 4 0.287 0.072 T o t a l 5 0.369 Ta b l e 3. 13a: A n a l y s i s o f Va r i a n c e f o r S o i l pH: S o i l 4 Sum of Mean F - r a t i o P r o b a b i l i t y Source DF squares square S t e r i l i z . 1 12.760 12.760 0.069 0.805 E r r o r 4 735.417 183.854 T o t a l 5 748.177 Table 3.13b: A n a l y s i s of V a r i a n c e f o r S o i l P: S o i l 4 Non S t e r i l e S t e r i l e PH P (ppm) 6.3 a* 85. 0 a 6.5 a 82.0 a * same l e t t e r w i t h i n i n category i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e s (a = 0.05 Tukey's (HSD) t e s t ) T a b l e 3.13c: S o i l pH and P (Olsen Method) b e f o r e and a f t e r S t e r i l i z a t i o n : S o i l 4 67 30 -i CT Hon Air Dried A1 A2 PA SOIL TREATMENT EM • Air Dried Figure 3.10: Mean Shoot Height: Soil 5 * same letter indicates no significant difference (a = 0.05 Tukey's (HSD) test) Source DF Sum of squares Mean square F-ratio Probability Air-Dried 1 35.112 351.120 1.508 0.224 S t e r i l i z . 4 1280.300 320.070 13.748 0. 000 Air-Dried 4 595.210 148.800 6. 392 0. 000 S t e r i l i z . Error 70 1629.600 23 .281 Total 79 3540.200 Table 3.14: Analysis of Variance for Shoot Height: Soil 5 68 weight compared t o the 2 a u t o c l a v i n g treatments ( F i g u r e 3.11). VAM Fungi Percent c o l o n i z a t i o n i n S o i l 5 was v e r y low (Table 3.16b) Mycorrhizae were pre s e n t i n the a i r - d r i e d c o n t r o l s o n l y . S o i l Analysis A i r - d r y i n g the s o i l d i d not s i g n i f i c a n t l y a f f e c t the pH of S o i l 5 but i t d i d i n c r e a s e the s o i l P (Table 3.17 and 3.18). S t e r i l i z i n g the s o i l caused s i g n i f i c a n t changes i n both the pH and P l e v e l s (Table 3.17 and 3.18). A u t o c l a v i n g the s o i l d i d not s i g n i f i c a n t l y a f f e c t the s o i l pH and P i n S o i l 5 ( F i g u r e 3.12 and 3 . 13). P a s t e u r i z i n g and a d d i n g f o r m a l i n t o t h e s o i l s i g n i f i c a n t l y i n c r e a s e d the pH e s p e c i a l l y i f the s o i l was a i r -d r i e d . Phosphorus was i n c r e a s e d s i g n i f i c a n t l y i n the p a s t e u r i z e d and f o r m a l i n t r e a t m e n t s . Though t h i s i n c r e a s e was not as g r e a t i n the a i r - d r i e d s o i l . VAM FUNGI AND PLANT GROWTH S t e r i l i z i n g the s o i l e l i m i n a t e s the mycorrhizae as expected. However, the g r e a t e s t shoot h e i g h t i n ARD s o i l s , are found i n the non or low m y c o r r h i z a l p l a n t s ( F i g u r e 3.14, 3.15 and 3.16). In n o n - s t e r i l i z e d s o i l , the g r e a t e r the m y c o r r h i z a l c o l o n i z a t i o n , the lower the p l a n t h e i g h t . DISCUSSION Based on pot t e s t s , a l l 5 s o i l s used i n t h i s study were p o s i t i v e l y i d e n t i f i e d as having ARD. While ARD s o i l s are i d e n t i f i e d by the i n c r e a s e i n p l a n t h e i g h t a f t e r s t e r i l i z a t i o n , S l y k h u i s and L i (1985) propose t h a t a s o i l f e r t i l i t y f a c t o r 69 E UJ o o cc 0.50 -, 0.40 -0.30 -£ 0.20 a,b* a a :1 n m l i i a,b CT Non Air Dried A1 A2 SOIL TREATMENT PA FM • Air Dried F i g u r e 3.11: Mean Root Dry Weight: S o i l 5 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e f o r mean of each s t e r i l i t y treatment (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y A i r - D r i e d 1 0.003 0.003 0. 598 0.442 S t e r i l i t y 4 0.106 0. 027 5.211 0. 001 A i r - D r i e d * 4 0. 036 0.009 1.785 0.142 S t e r i l i t y E r r o r 70 0. 357 0. 005 T o t a l 79 0. 503 Table 3.15: A n a l y s i s o f V a r i a n c e f o r Root Dry Weight: S o i l 5 70 Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y A i r - D r i e d 1 68.543 68.543 2.420 0.124 S t e r i l i t y 4 441.706 110.427 3 .899 0. 006 A i r - D r i e d * 4 274.170 68.543 2.420 0. 056 S t e r i l i t y E r r o r 70 1982.568 28.322 T o t a l 79 2766.987 Ta b l e 3.16a: A n a l y s i s o f V a r i a n c e f o r Mycor r h i z a e : S o i l 5 Non A i r D r i e d A i r D r i e d C o n t r o l 0.00 a* 10.50 b Au t o c l a v e 1 0.00 a 0. 00 a Au t o c l a v e 2 0.00 a 0.00 a P a s t e u r i z e 0. 00 a 0. 00 a Formal i n 0.00 a 0.00 a Tabl e 3.16b: Percent M y c o r r h i z a l C o l o n i z a t i o n : S o i l 5 same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e s (a =0.05 Tukey's (HSD) t e s t ) 71 CT A l A2 PA FM SOIL TREATMENT • Non Air Dried • Air Dried F i g u r e 3.12: S o i l pH: S o i l 5 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y A i r - D r i e d S t e r i l i t y A i r - D r i e d S t e r i l i t y 1 4 4 0.0003 1.2230 0.7180 0.0003 0.3058 0.1795 0.011 10.194 5.983 0.917 0. 000 0. 002 E r r o r T o t a l 20 29 0.6000 2.5417 0.0300 Table 3.17: A n a l y s i s o f V a r i a n c e f o r S o i l pH: S o i l 5 72 200 - i CT A1 A2 PA FM SOIL TREATMENT • Non Air Dried • Air Dried F i g u r e 3.13: S o i l Phosphorus (Olsen Method): S o i l 5 *. same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y A i r - D r i e d 1 991.87 991.87 4.19 0.054 S t e r i l i t y 4 22393.00 5598.30 23.66 0. 000 A i r - D r i e d * 4 6444.10 1611.00 6.80 0.001 S t e r i l i t y E r r o r 20 4732.30 236.61 T o t a l 29 34561.00 Table 3.18: A n a l y s i s of V a r i a n c e f o r S o i l P: S o i l 5 73 PLANT HEIGHT (cm) 60 -, • Plant Height • Percent Colonization PERCENT COLONIZATION Control 11-55-0 34-0-0 0-45-0 Sterile Sterile + Sterife + Sterile + 11-55-0 34-0-0 0-45-0 TREATMENT Figure 3.14: Plant Height and VA Mycorrhizal Colonization: S o i l 1 PLANT HEIGHT (cm) PERCENT COLONIZATION Control 11-55-0 34-0-0 0-45-0 Sterile TREATMENT 11-55-0 34-0-0 0-45-0 Figure 3.15: Plant Height and VA Mycorrhizal Colonization: S o i l 2 74 PLANT HEIGHT (cm) 30 PERCENT COLONIZATION 30 • Plant Height • Root Colonization AD AD + A2 AD + FM NAD + Al NAD + PA AD + Al AD + PA NAD NAD + A2 NAD + FM TREATMENT Figure 3.16: Pl a n t Height and VA M y c o r r h i z a l C o l o n i z a t i o n : S o i l 5 75 e s p e c i a l l y . a combination of N and P may be i n v o l v e d i n ARD. T h i s study concurs w i t h t h e i r p r o p o s a l . N i t r o g e n alone may a l s o be i n v o l v e d i n ARD, though P alone i s n o t . P alone may even be d e l e t e r i o u s t o shoot growth i n ARD s o i l s . These f i n d i n g s support the recommendations t o growers t o s t e r i l i z e and add 11-55-0 f e r t i l i z e r s t o o l d apple s o i l s . While shoot h e i g h t i s u s u a l l y measured as an i n d i c a t i o n of good p l a n t growth, the importance of r o o t growth i s o f t e n ignored or minimized. In ARD s o i l s N and the combination of N and P do not a f f e c t r o o t mass. While P may have no e f f e c t on shoot h e i g h t , i t does i n c r e a s e r o o t mass. In t h i s case the p l a n t appears t o be maximizing r o o t growth at the expense of shoot growth. T h i s may b e n e f i t the young t r e e i n the f i r s t y ear of growth a f t e r t r a n s p l a n t i n g and may l e a d t o a h e a l t h i e r t r e e i n subsequent y e a r s . The n u t r i e n t s t a t u s of ARD s o i l s a f t e r s t e r i l i z a t i o n i s u s u a l l y i g n o r e d . The lowering of pH a f t e r a u t o c l a v i n g suggests t h a t o r g a n i c a c i d s are being r e l e a s e d . The i n c r e a s e i n P a f t e r s t e r i l i z i n g i n d i c a t e s t h a t m i c r o b i a l and o r g a n i c matter P i s b e i n g r e l e a s e d a f t e r treatment. Though these changes may not be of s u f f i c i e n t magnitude to a f f e c t apple growth. A i r - d r y i n g the s o i l does not a f f e c t pot b i o a s s a y s f o r ARD. I t i s t h e r e f o r e not necessary t o recommend t o growers t h a t they keep t h e i r s o i l moist p r i o r t o doing an ARD t e s t . In the absence of a host p l a n t , d r y i n g the s o i l appears t o p r e s e r v e v i a b l e VAM p r o p a g u l e s . Drying the s o i l i s not recommended as an a l t e r n a t i v e t o a l l e v i a t e ARD i n the o r c h a r d . Pot t e s t s however, show t h a t 76 d r y i n g the s o i l combined wi t h p a s t e u r i z i n g o r adding f o r m a l i n causes marked i n c r e a s e s i n shoot h e i g h t . T h i s treatment c o u l d be a v i a b l e a l t e r n a t i v e t o growers and should be f u r t h e r t e s t e d i n f i e l d t r i a l s . VA mycorrhizae have been proposed as p l a y i n g a r o l e i n the ARD problem i n B.C. Phosphorus and N f e r t i l i z e r s alone do not harm these m u t u a l i s t i c f u n g i , s u g g e s t i n g t h a t these f e r t i l i z e r s may not be r e a d i l y a v a i l a b l e t o the p l a n t . However, the combinations of the two does, s u g g e s t i n g t h a t t h i s f e r t i l i z e r may be immediately a v a i l a b l e t o the p l a n t , thereby e l i m i n a t i n g the need f o r the VAM a s s o c i a t i o n . The r e l a t i o n s h i p between VAM fungus and host shown i n t h i s study goes c o n t r a r y t o the u s u a l m u t a l i s t i c a s s o c i a t i o n between the host and fungus. In ARD s o i l s , the p l a n t may be under so much s t r e s s t h a t the fungus may be behaving p a r a s i t i c a l l y . The fungus i n these s o i l s may be c a u s i n g a d e l e t e r i o u s carbon d r a i n on the p l a n t m a n i f e s t e d by t h e d e c r e a s e i n p l a n t h e i g h t . The exchangeable P t h a t the p l a n t r e c e i v e s may not be enough to overcome t h i s carbon d r a i n . T h i s r e l a t i o n s h i p between m y c o r r h i z a l fungus and apple host i n ARD s o i l s must be f u r t h e r i n v e s t i g a t e d . The e l u c i d a t i o n of s o i l n u t r i e n t s i n ARD s o i l s should be f u r t h e r i n v e s t i g a t e d as w e l l . The l e v e l s o f s o i l n u t r i e n t s b e f o r e and a f t e r s t e r i l i z a t i o n and i t s e f f e c t on both s o i l micro- and macro- fauna should be f u r t h e r i n v e s t i g a t e d . The ARD c a u s a l agent may be d e t r i m e n t a l l y e f f e c t e d by s o i l n u t r i e n t changes a f t e r s t e r i l i z a t i o n . APPLE GROWTH IN APPLE REPLANT DISEASED SOILS AFTER INOCULATION WITH VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGI INTRODUCTION Apple r e p l a n t d i s e a s e (ARD) can be overcome by fumigating the s o i l p r i o r t o t r a n s p l a n t i n g (Mai and Abawi 1981, Sewell and White 1979, S l y k h u i s and L i 1985). The symptoms of the d i s e a s e are ambiguous, the key f a c t o r being poor growth, i n d i c a t i v e of a r o o t problem. While the d i s e a s e i s b i o l o g i c a l i n n a t u r e , no s i n g l e known pathogen has been i m p l i c a t e d i n the d i s e a s e . Apple s e e d l i n g s w i l l a l s o respond p o s i t i v e l y t o the a d d i t i o n of a n i t r o g e n and phosphorus f e r t i l i z e r a p p l i e d a f t e r s o i l f umigation ( S l y k h u i s and L i 1985). T h i s growth response o f apple to f u m i g a t i o n and f e r t i l i z e r t r e a t m e n t s , suggests t h a t v e s i c u l a r -a r b u s c u l a r m y c o r r h i z a l (VAM) f u n g i may be i m p l i c a t e d i n ARD. F r u i t t r e e s have h i g h m y c o r r h i z a l dependencies. M y c o r r h i z a l avocado t r e e s have been shown t o not only grow f a s t e r than non m y c o r r h i z a l t r e e s but the a b i l i t y of the m y c o r r h i z a l t r e e t o s u r v i v e t r a n s p l a n t i n g surpasses t h a t of nonmycorrhizal t r e e s (Menge e t a l . 1978b) . Increases i n p l a n t growth due t o m y c o r r h i z a l f u n g i i n both greenhouse and f i e l d t r i a l s have been demonstrated f o r c i t r u s as w e l l (Hattingh and Gerdemann 1975). Apples form mycorrhizae wi t h a wide range of VAM fungal s p e c i e s (Benson and Covey 1976, Hoepfner e t a l . 1983, M i l l e r et a l . 1985b, P l e n c h e t t e e t a l . 1982, R e i c h 1988). D i f f e r e n t VAM s p e c i e s induce d i f f e r e n t growth responses i n a p p l e s . G e n e r a l l y , t o t a l a p p l e growth i s enhanced due t o the presence of 78 mycorrhizae. Mosse (1957), and M i l l e r et a l . (1985b) found t h a t n o n m y c o r r h i z a l a p p l e p l a n t s outgrew or grew as w e l l as m y c o r r h i z a l p l a n t s i n i t i a l l y , but a f t e r 8 weeks the m y c o r r h i z a l s e e d l i n g s continued t o grow, whereas the nonmycorrhizal seedlings had s e t t e r m i n a l buds. E v e n t u a l l y the m y c o r r h i z a l p l a n t s had g r e a t e r r o o t , stem and l e a f growth compared to the nonmycorrhizal p l a n t s . I n o c u l a t i o n w i t h Glomus f a s c i c u l a t u m (Thaxter) Gerdemann and Trappe emend. Walker and Koske r e s u l t e d i n b e t t e r growth of apple s e e d l i n g s than i n o c u l a t i o n w i t h G. mosseae (Nicolson and Gerdemann) Gerdemann and Trappe (Benson and Covey 1976). However, apples i n o c u l a t e d w i t h G. microcarpum Tulasne and Tulasne showed l i t t l e i n c r ease i n p l a n t growth compared t o the nonmycorrhizal c o n t r o l s (Covey et a l . 1981). M i l l e r et a l . (1985b) and Plenchette et a l . (1982) found s i m i l a r trends w i t h the d i f f e r e n t VAM species used t o i n o c u l a t e apple s e e d l i n g s . Plenchette et a l . (1982) a l s o found t h a t percent m y c o r r h i z a l c o l o n i z a t i o n c o r r e l a t e d d i r e c t l y t o p l a n t height. However, Reich (1988) found t h a t w h i l e G. i n t r a r a d i c e s Schenck and Smith r e s u l t e d i n the highest l e v e l s of m y c o r r h i z a l c o l o n i z a t i o n , apples i n o c u l a t e d w i t h G. epicfaeum Daniels and Trappe (= Glomus  versiforme (Karsten) Berch) r e s u l t e d i n the g r e a t e s t p l a n t growth. I t was not the r a t e of c o l o n i z a t i o n t h a t c o r r e l a t e d d i r e c t l y t o p l a n t growth, but r a t h e r the amounts of root P. This v a r i a t i o n i n apple response t o VAM i n o c u l a t i o n i s t hought t o be a r e s u l t of the discrepancy between the 79 e x t r a m a t r i c a l hyphae found o u t s i d e the r o o t and the i n t r a m a t r i c a l hyphae found i n s i d e the r o o t . The i n f l u e n c e of VAM f u n g i may not be d i r e c t l y r e l a t e d t o the i n t r a m a t r i c a l hyphae but r a t h e r due t o the e x t r a m a t r i c a l hyphal ex t e n s i o n s ( M i l l e r e t a l . 1985b). Another reason f o r the d i f f e r e n t responses t o VAM f u n g i i s the amount of a v a i l a b l e P i n the s o i l . The uptake of P i s not i n c r e a s e d by mycorrhizae i n the presence of adequate P. However, apple growth i s improved due t o mycorrhizae i n s o i l s low i n P. Hoepfner et a l . (1983) have shown t h a t apples grown i n fumigated s o i l s r e q u i r e d lower P supplements f o r optimal growth as long as the s o i l was i n o c u l a t e d w i t h VAM f u n g i . At h i g h e r l e v e l s of P, the m y c o r r h i z a l c o l o n i z a t i o n was decreased d r a m a t i c a l l y . They suggest t h a t fumigating s o i l s f o r apple p r o d u c t i o n should be f o l l o w e d by e i t h e r P f e r t i l i z e r s or i n o c u l a t i o n s of VAM f u n g i . While apples respond w e l l t o VAM f u n g a l i n o c u l a t i o n s , l i t t l e work has been done on the growth of i n o c u l a t e d apples i n ARD s o i l s . Utkhede (1987) demonstrated t h a t under greenhouse c o n d i t i o n s , apples i n o c u l a t e d with Glomus spp. grew s i g n i f i c a n t l y b e t t e r i n ARD s o i l s t h a t were not p a s t e u r i z e d as w e l l as i n s o i l s t h a t were. S i m i l a r r e s u l t s were found i n apple s e e d l i n g s grown on l y i n s t e r i l i z e d ARD s o i l s i n o c u l a t e d w i t h VAM f u n g i (Hoepfner e t a l . 1983, Sewell and Roberts 1984,1985). However, i n n o n s t e r i l i z e d s o i l , VAM f u n g i d i d not enhance apple growth (Sewell and Roberts 1984) . VAM i n o c u l a t i o n s of o l d a l f a l f a s o i l s overcame ' a l f a l f a s i c k n e s s ' and i t i s suggested t h a t the indigenous VAM f u n g i are 80 not as e f f e c t i v e as the i n o c u l a t e d f u n g i i n r o o t i n f e c t i o n and s t i m u l a t i o n o f p l a n t growth (Hwang 1988). The o b j e c t i v e s o f t h i s study were t o determine i f i n o c u l a t i n g ARD s o i l s with d i f f e r e n t VAM f u n g i would overcome ARD; and t o compare the f e r t i l i z e r 11-55-0, used i n standard pot b i o a s s a y s , w i t h VAM f u n g i i n s e e d l i n g s response grown i n ARD s o i l s . MATERIAL AND METHODS THE SOILS S o i l 2 and S o i l 5 which were p r e v i o u s l y i d e n t i f i e d as having ARD were used i n t h i s s tudy. S o i l 2 was s t e r i l i z e d and S o i l 5 was p a s t e u r i z e d as above (Chapter 3, pages 50 and 52). THE FUNGI Both s o i l s were i n o c u l a t e d w i t h t h r e e pure VAM fu n g a l s p e c i e s , Glomus clarum N i c h o l s o n and Schenck (GC) (1.4 X 103 l i v e propagules per m l ) , Glomus i n t r a r a d i c e s (GI) , (3.3 X 102 l i v e propagules per ml) and Glomus v e r s i f o r m e (Karsten) Berch (GV) , (1.4 X 103 l i v e propagules per ml) which were obt a i n e d from Premier Peat, Rivere-du-Loup, Quebec. S o i l 5 was a l s o i n o c u l a t e d w i t h a G. monosporum Gerdemann and Trappe (OR), (1.4 X 102 l i v e propagules per ml) from the U n i v e r s i t y of B.C. r e s e a r c h farm at O y s t e r R i v e r , B.C. C o l o n i z e d onion r o o t fragments p l u s i n o c u l a t e d T u r f a c e were used as the inoculum source f o r the 3 s p e c i e s from Quebec, and d r i e d T u r f a c e c o n t a i n i n g l i v e spores were used as the inoculum source f o r the OR s p e c i e s . INOCULATING SOILS WITH VAM FUNGI 81 S t e r i l i z e d and n o n s t e r i l i z e d s o i l were i n o c u l a t e d w i t h the f o l l o w i n g r a t e s o f VAM f u n g i : OR, 133 mls/1 s o i l ; GI, 57 mls/1 s o i l ; both GC and GV, 13 mls/1 s o i l . Ammonium phosphate f e r t i l i z e r (11-55-0) at 1.5 gm/1 (0.165 gm N, 0.36 gm P) s o i l was gi v e n as a f i f t h t reatment. There was a l s o an u n t r e a t e d c o n t r o l (CT) . The OR s p e c i e s was used w i t h S o i l 5 o n l y . A l l treatments were r e p l i c a t e d 6 t i m e s . SEEDLING GROWTH AND ANALYSIS One 1 - 2 l e a f stage apple s e e d l i n g was t r a n s p l a n t e d i n t o each pot and then grown at 24°C on a p l a n t bench. A f t e r t e n weeks, the h e i g h t and shoot dry weight were recorded f o r a l l s e e d l i n g s . The r o o t s were f i x e d i n FAA f o r VAM a n a l y s i s (see Chapter 2, pp 23). Root dry weights were taken a f t e r VAM a n a l y s i s . A l l r o o t fragments were p l a c e d on f i l t e r p a p e r s, washed r e p e a t e d l y t o e l i m i n a t e the l a c t i c a c i d and then oven-dried at 60°C f o r 5 days. VAM c o l o n i z a t i o n was determined as p r e v i o u s l y d e s c r i b e d (Chapter 2, pp 23). RESULTS SHOOT HEIGHT In S o i l 2 shoot h e i g h t i n c r e a s e d s i g n i f i c a n t l y due t o s t e r i l i z a t i o n (Table 4.1). S i m i l a r responses were found i n S o i l 5 due t o p a s t e u r i z a t i o n (Table 4.2). The f e r t i l i z e r treatment r e s u l t e d i n s i g n i f i c a n t i n c r e a s e s i n shoot h e i g h t i n both s t e r i l i z e d and n o n s t e r i l i z e d S o i l 2 and i n s t e r i l i z e d S o i l 5 (F i g u r e s 4.1 and 4.2). In S o i l 5 p a s t e u r i z i n g and adding f e r t i l i z e r r e s u l t e d i n an a d d i t i v e 82 GC GV GI 11 - 5 5 - 0 CT Non Sterilized TREATMENT • Sterilized F i g u r e 4.1: Shoot Height at 10 Weeks: S o i l 2 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e f o r mean of each treatment (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y S t e r i l i t y 1 172.110 172.110 11.784 0. 001 Treatment 4 1131.800 282.950 19.373 0. 000 S t e r i l i t y * 4 123 . 740 30.935 2.118 0. 093 Treatment E r r o r 49 715.670 14.605 T o t a l 58 2114.000 Table 4.1: A n a l y s i s o f V a r i a n c e f o r Shoot Height: S o i l 2 83 i n c r e a s e i n shoot h e i g h t . No s i g n i f i c a n t d i f f e r e n c e s o c c u r r e d among any of the othe r treatments i n S o i l 2 ( F i g u r e 4.1). In S o i l 5, any s i g n i f i c a n t i n c r e a s e s i n shoot h e i g h t o c c u r r e d o n l y i f the s o i l was p a s t e u r i z e d . In p a s t e u r i z e d S o i l 5, the GV s p e c i e s i n c r e a s e d shoot h e i g h t s i g n i f i c a n t l y compared t o the GC treatment o n l y ( F i g u r e 4.2). The remaining i n o c u l a t i o n s d i d not d i f f e r s i g n i f i c a n t l y from each o t h e r . SHOOT DRY WEIGHT P a t t e r n s o f shoot dry weight i n both s o i l s f o l l o w e d shoot h e i g h t c l o s e l y , i n c r e a s i n g s i g n i f i c a n t l y when the s o i l was s t e r i l i z e d o r p a s t e u r i z e d (Table 4.3 and 4.4). T r e a t i n g S o i l 2 wit h VAM f u n g i d i d not r e s u l t i n any changes i n shoot dry weight even i f the s o i l was s t e r i l i z e d ( F i g u r e 4.3). The f e r t i l i z e r 11-55-0 however, i n c r e a s e d shoot dry weight i n both s t e r i l e and n o n s t e r i l e s o i l s . In S o i l 5 t h e r e was an i n c r e a s e i n shoot dry weight i n the GV, GI and 11-55-0 treatments only a f t e r p a s t e u r i z a t i o n ( F i g u r e 4.4), though t h i s e f f e c t was e s p e c i a l l y s t r i k i n g i n the 11-55-0 treatment. Root Dry Weight Root dry weight i n c r e a s e d s i g n i f i c a n t l y due t o s t e r i l i z a t i o n i n S o i l 2 (Table 4.5) and p a s t e u r i z a t i o n i n S o i l 5 (Table 4.6). However, i f S o i l 2 was s t e r i l i z e d and g i v e n 11-55-0 a r e d u c t i o n i n r o o t growth o c c u r r r e d . In n o n p a s t e u r i z e d S o i l 5, none of the treatments a f f e c t e d the r o o t growth ( F i g u r e 4.6). However, a s i g n i f i c a n t i n t e r a c t i o n 84 GC GV INcm Pasteurized GI OK 11-55-0 CT TREATMENT • Pasteurized Figure 4.2: Shoot Height at 10 Weeks: S o i l 5 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) Sum of Mean F-- r a t i o P r o b a b i l i t y Source DF squares square S t e r i l i t y 1 1645.900 1645.900 146.760 0.000 Treatment 5 556.460 111.290 9.924 0.000 S t e r i l i t y * 5 770.770 154.150 13.746 0.000 Treatment E r r o r 59 661.670 11.215 T o t a l 70 3629.400 Table 4.2: A n a l y s i s of Variance f o r Shoot Height: S o i l 5 85 E a: o >-an a o o •r. tn GC GV GI 11 - 5 5 - 0 CT TREATMENT Non Sterilized • Sterilized F i g u r e 4.3: Shoot Dry Weight at 10 Weeks: S o i l 2 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean square F - r a t i o P r o b a b i l i t y S t e r i l i t y 1 3.113 3.113 29.341 0. 000 Treatment 4 17.295 4.324 40.756 0.000 S t e r i l i t y * 4 1.566 0.392 3.691 0. 011 Treatment E r r o r 49 5.198 0.106 j T o t a l 58 26.680 Table 4.3: A n a l y s i s of Variance f o r Shoot Dry Weight: S o i l 2 86 GC GV GI OR TREATMENT 11-55 -0 CT I Non Pasteurized • Pasteurized F i g u r e 4.4: Shoot Dry Weight a t 10 Weeks: S o i l 5 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) Sum of Mean F - r a t i o P r o b a b i l i t y Source DF squares square S t e r i l i t y 1 19.467 19.466 219.140 0.000 Treatment 5 3 .453 0. 691 7.775 0.000 S t e r i l i t y * 5 4.815 0.963 10.840 0.000 Treatment E r r o r 59 5.241 0. 089 T o t a l 70 32.963 Table 4.4: A n a l y s i s of V a r i a n c e f o r Shoot Dry Weight: S o i l 5 87 0.30 -GC GV GI 11-55-0 CT TREATMENT • Non Sterilized • Sterilized Figure 4.5: Root Dry Weight at 10 Weeks: S o i l 2 * same l e t t e r indicates no s i g n i f i c a n t difference (a = 0.05 Tukey's (HSD) test) Source DF Sum of squares Mean square F- r a t i o P r o b a b i l i t y S t e r i l i t y 1 0.187 0.187 30.355 0. 000 Treatment 4 0. 027 0. 007 1.109 0. 363 S t e r i l i t y * 4 0.151 0.038 6.139 0. 000 Treatment Error 49 0. 302 0. 006 Total 58 0.666 Table 4.5: Analysis of Variance for Root Dry Weight: S o i l 88 between p a s t e u r i z i n g t h i s s o i l and adding the f u n g i GV and GI o c c u r r e d . Root dry weight i n c r e a s e d s i g n i f i c a n t l y i n these t r e a t m e n t s . VAM Fungi S t e r i l i z i n g S o i l 2 d i d n o t a f f e c t t h e m y c o r r h i z a l c o l o n i z a t i o n o f r o o t s (Table 4.7), but i t d i d i n S o i l 5 (Table 4.8). There were s i g n i f i c a n t i n t e r a c t i o n s between s t e r i l i z a t i o n and treatment f o r both s o i l s . In S o i l 2 the GV, i n s t e r i l i z e d s o i l s and the GI, i n both s t e r i l i z e d and n o n s t e r i l i z e d s o i l s i n c r e a s e d r o o t c o l o n i z a t i o n s i g n i f i c a n t l y compared t o the 11-55-0 treatment and the s t e r i l i z e d c o n t r o l ( F i g u r e 4.7). No d i f f e r e n c e i n m y c o r r h i z a l c o l o n i z a t i o n o c u r r e d i n t h i s s o i l among the v a r i o u s f u n g i . The VAM c o l o n i z a t i o n was not a f f e c t e d i n s t e r i l i z e d s o i l s i f they were r e - i n o c u l a t e d . S t e r i l i z i n g t h i s s o i l o r adding 11-55-0 s i g n i f i c a n t l y decreased VAM f u n g i . S o i l 5 had a low indigenous p o p u l a t i o n o f VAM f u n g i . In t h i s s o i l the GC i n c r e a s e d m y c o r r h i z a l c o l o n i z a t i o n even i n the non p a s t e u r i z e d s o i l s ( F i g u r e 4.8). There were s i g n i f i c a n t i n c r e a s e s i n VAM c o l o n i z a t i o n among the other treatments o n l y i f the s o i l was p a s t e u r i z e d . Though the v a r i o u s VAM f u n g i d i d not d i f f e r s i g n i f i c a n t l y from each o t h e r . The GI was the be s t c o l o n i z e r i n the p a s t e u r i z e d s o i l o n l y . There were s i g n i f i c a n t i n c r e a s e s i n VAM c o l o n i z a t i o n with the OR and GI a f t e r p a s t e u r i z a t i o n . VAM FUNGI AND PLANT GROWTH In both ARD s o i l s used i n t h i s study, VAM f u n g i d i d not 89 X o >-Q o o 0.50-1 0.40 -0.30 -* 0.20 0.10 -0.00 "C3 1 o • •o a o jQ O GC GV Non Pasteurized GI OR TREATMENT 1 1 - 5 5 - 0 CT • Pasteurized Figure 4.6: Root Dry Weight at 10 Weeks: Soil 5 * same letter indicates no significant difference (a = 0.05 Tukey's (HSD) test) Source DF Sum of squares Mean square F-ratio Probability S t e r i l i t y 1 0. 530 0. 530 92.001 0. 000 Treatment 5 0.112 0. 022 3.892 0. 004 S t e r i l i t y * 5 0.089 0. 018 3 .106 0.015 Treatment Error 59 0.340 0.006 Total 70 1.079 Table 4.6: Analysis of Variance for Root Dry Weight: Soil 90 o M rz. o - j o o UJ o GC Hon Sterilized GV GI 11-55-0 CT TREATMENT • Sterilized F i g u r e 4.7: Percent M y c o r r h i z a l C o l o n i z a t i o n : S o i l 2 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean F-square • r a t i o P r o b a b i l i t y S t e r i l i t y 1 1121.200 1121.200 3.089 0.085 Treatment 4 24354.000 6088.500 16.775 0. 000 S t e r i l i t y * 4 11261.000 2815.200 7.756 0.000 Treatment E r r o r 49 17785.000 362.960 T o t a l 58 54239.000 Ta b l e 4.7: A n a l y s i s of Va r i a n c e f o r Mycorrhizae: S o i l 2 91 GC GV GI OR 11-55-0 CT TREATMENT • Non Pasteurized • Pasteurized F i g u r e 4.8: Percent C o l o n i z a t i o n : S o i l 5 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) Source DF Sum of squares Mean F-square - r a t i o P r o b a b i l i t y S t e r i l i t y 1 6590.600 6590.600 31.584 0.000 Treatment 5 19293.000 3858.600 18.491 0. 000 S t e r i l i t y * 5 6690.400 1338.100 6.412 0. 000 Treatment E r r o r 59 12312.000 208.670 T o t a l 70 45255.000 Table 4.8: A n a l y s i s o f V a r i a n c e f o r Mycorrhizae: S o i l 5 92 was not s t e r i l i z e d or p a s t e u r i z e d c o n c u r r i n g w i t h Sewell and Roberts (1984,1985) (F i g u r e 4.9 and 4.10). While t h e r e was no l i n e a r c o r r e l a t i o n between m y c o r r h i z a l c o l o n i z a t i o n and shoot or r o o t growth, i t appears as i f those p l a n t s w i t h the lowest VAM c o l o n i z a t i o n show the g r e a t e s t shoot growth i n n o n s t e r i l i z e d ARD s o i l ( F i g u r e 4.9). I t may be t h a t the VAM f u n g i are behaving as pathogens i n n o n s t e r i l i z e d ARD s o i l s . In ARD pot b i o a s s a y s , s o i l s t r e a t e d with the f e r t i l i z e r 11-55-0 gave the g r e a t e s t i n c r e a s e i n shoot growth compared t o any VAM i n o c u l a t i o n s ( F i g u r e s 4.9 and 4.10). T h i s f e r t i l i z e r however, caused d r a s t i c r e d u c t i o n s i n the VAM c o l o n i z a t i o n i n s t e r i l i z e d and non s t e r i l i z e d s o i l s . While the v a r i o u s f u n g i d i d not induce as g r e a t a response i n shoot growth as the f e r t i l i z e r treatment, they induced the g r e a t e s t r o o t growth ( F i g u r e s 4.11 and 4.12). There i s an o p p o s i t e response of s e e d l i n g s t o f e r t i l i z e r except i n the n o n s t e r i l e treatment of S o i l 2. DISCUSSION The growth responses t h a t o c c u r r e d i n i n o c u l a t e d m y c o r r h i z a l apple p l a n t s were unexpected. In both ARD s o i l s used i n t h i s s t u d y , VAM f u n g i d i d not induce the expected p l a n t growth i n c r e a s e s i n pot b i o a s s a y s i f the s o i l was not s t e r i l i z e d or p a s t e u r i z e d c o n c u r r i n g with Sewell and Roberts (1984,1985) ( F i g u r e 4.9 and 4.10). T h i s goes c o n t r a r y t o Utkhede's (1987) stu d y . Utkhede (1987) used a mixture of Glomus spp. a t 1:1 ( v o l / v o l ) i n o c u l u m : s o i l . I t may be t h a t i n ARD s o i l s VAM PLANT HEIGHT (cm) GC Plant Height GV GI 11-55-0 TREATMENT C T PERCENT COLONIZATION - 100 • Root Colonization Figure 4.9: Mycorrhizal Colonization and Plant Height: S o i l 2 94 PLANT HEIGHT (cm) GC GV Plant Height PERCENT COLONIZATION Non Pasteurized Pasteurized a GI OR 1 1 - 5 5 - 0 CT TREATMENT O Root Colonization Figure 4.10: Mycorrhizal Colonization and Plant Height: Soil 5 95 ROOT DRY WEIGHT (gm) 0.40 -0.30 -Non Sterile Sterile PERCENT COLONIZATION GV GI 11-55-0 CT Root Dry Weight TREATMENT a Root Colonization Figure 4.11: Mycorrhizal Colonization and Root Dry Weight: S o i l 2 ROOT DRY WEIGHT (gm) 0.50 0.40 H 0.30 0.20 0.10 H 0.50 -0.40 -0.30 H 0.20 A 0.10 H 0.00 Non Pasteurized Pasteurized GC GV Root Dry Weight PERCENT COLONIZATION - 100 - 80 - 60 [- 40 20 "100 80 - 60 40 20 GI OR 11-55-0 CT TREATMENT <=• Root Colonization Figure 4.12: M y c o r r h i z a l C o l o n i z a t i o n and Root Dry Weight: S o i l 5 97 propogules must surpass a t h r e s h o l d l e v e l i n order t o induce p l a n t growth i n c r e a s e s . In t h i s study, as i n Sewell (1985), pure c u l t u r e s of VAM f u n g i were used, e l i m i n a t i n g any other microbe e f f e c t s . I t may be t h a t i n Utkhede's (1987) study the inoculum may have had other p l a n t growth promoting micro-organisms. Mosse (1957) i n d i c a t e d t h a t m y c o r r h i z a l apples are slow growers t h a t w i l l a f t e r 8 weeks outgrow nonmycorrhizal p l a n t s . This study was terminated a f t e r 10 weeks and t h i s may have been too soon to see the e f f e c t s of the VAM f u n g i on shoot growth i n ARD s o i l s . I t appears t h a t i n o c u l a t e d apples w i l l f i r s t b u i l d a s o l i d root mass which may e v e n t u a l l y lead t o h e a l t h i e r shoot growth. ARD s o i l s u s u a l l y have indigenous m y c o r r h i z a l f u n g i and these f u n g i are not e f f e c t i v e i n s t i m u l a t i n g p l a n t growth. The ARD f a c t o r , which appears to be i n h i b i t i n g the e f f e c t i v e n e s s of these f u n g i , appears t o be i n h i b i t i n g the i n o c u l a t e d f u n g i i n n o n s t e r i l i z e d ARD s o i l s as w e l l . There are predatory microfauna i n s o i l s t h a t l i m i t the growth of VAM f u n g i . The collembola Folsomia C a n d i d a feeds on the e x t e r n a l hyphae of VAM f u n g i , thereby reducing the e f f e c t i v e n e s s of the fungus (Warnock et a l . 1982) . The feeding a c t i v i t y of predatory microfauna may e x p l a i n why VAM i n o c u l a t i o n s d i d not induce p l a n t growth increases i n n o n s t e r i l i z e d ARD s o i l s , but d i d i n s t e r i l i z e d ARD s o i l s . I t may a l s o e x p l a i n why p l a n t s w i t h high l e v e l s of VAM c o l o n i z a t i o n do not induce high growth increases. I t i s p o s s i b l e t h a t the i n t r a m a t r i c a l hyphae i n p l a n t s grown i n 98 ARD s o i l s do not c o r r e l a t e with e x t r a m a t r i c a l hyphae which may be reduced by p r e d a t o r s . In the quest t o understand ARD, s o i l microfauna are i g n o r e d . I t i s p o s s i b l e , t h a t the microfauna, i n a c t i n g as p r e d a t o r s of VAM f u n g i , reduce the e x t r a m a t r i c a l hyphae and the e f f e c t i v e n e s s of the VAM fungus. The i n t e r a c t i o n s of the microfauna i n ARD s o i l s should be s t u d i e d f u r t h e r i n the ARD complex. B a y l i s (1967) documented t h a t i n cases of adequate s o i l P VAM i n o c u l a t i o n s w i l l cause a d e p r e s s i o n i n p l a n t growth. Both s o i l s used i n t h i s study had h i g h s o i l P. I t may be t h a t the l a c k of growth response of i n o c u l a t e d s e e d l i n g s t o VAM c o u l d have been a r e s u l t of the h i g h l e v e l s of s o i l P. Sewell (1984), proposes t h a t Pythium spp., as the c a u s a l agent of ARD, e x e r t t h e i r e f f e c t by d e s t r o y i n g the r o o t c o r t i c a l c e l l s and any subsequent r o o t growth i s s e v e r l y r e s t r i c t e d . Through the d e s t r u c t i o n of these c e l l s , the VAM a s s o c i a t i o n s are d e s t r o y e d . R e s u l t s from t h i s study i n d i c a t e t h a t t h i s i s not the c a s e . VAM c o l o n i z a t i o n i s h i g h i n p l a n t s a f f e c t e d by ARD. In f a c t i t appears t h a t the h i g h e r the VAM c o l o n i z a t i o n the lower the apple growth i n nontreated ARD s o i l s . I f a f a c u l t a t i v e pathogen such as Pythium or Fusarium i s i n t r o d u c e d w i t h a VAM fungus, the fungus may a i d i n or cause growth d e p r e s s i o n s , r a t h e r than induce growth i n c r e a s e s ( H a l l 1981). I t i s p o s s i b l e t h a t a f a c u l t a t i v e pathogen i s p r e s e n t i n these s o i l s , a f f e c t i n g the p l a n t growth i n such a manner t h a t the VAM f u n g i are unable t o overcome any pathogenic e f f e c t s . I f VAM c o l o n i z a t i o n goes beyond a c e r t a i n t h r e s h o l d , then they may a l s o behave as pathogens r a t h e r than symbionts. VAM f u n g i do not b e n e f i t t h e i r hosts i n ARD s o i l s and i n f a c t may behave as pathogens. The l a r g e r the VAM c o l o n i z a t i o n the more n u t r i e n t exchange s i t e s , and the more C i s taken from the h o s t . I f no or minimal amounts of P are being exchanged then the fungus may be c a u s i n g a d e l e t e r i o u s C d r a i n on the p l a n t . VAM f u n g i may e f f e c t i v e l y compete f o r photosynthates with t h e i r h o s t s (Buwalda et a l . 1982). C o l o n i z a t i o n o f r o o t s by VAM f u n g i l e a d s t o the decrease i n carbohydrates w i t h i n the shoots w h i l e not a f f e c t i n g carbohydrates i n the r o o t s . T h i s may e x p l a i n the r e s u l t s found i n t h i s s t u d y . I t i s p o s s i b l e t h a t apple s e e d l i n g s grown i n ARD s o i l s are under s u f f i c i e n t s t r e s s t h a t the VAM fungus e f f e c t i v e l y competes f o r carbohydrates and t h i s leads t o depressed growth e s p e c i a l l y i n s h o o t s . The f e r t i l i z e r 11-55-0, at the r a t e a p p l i e d , gave the g r e a t e s t i n c r e a s e i n p l a n t h e i g h t . T h i s f e r t i l i z e r appears t o be a v a i l a b l e f o r p l a n t use immediately or v e r y soon a f t e r a p p l i c a t i o n . From i t s e f f e c t on the VAM f u n g i , i t appears t h a t i t moves r a p i d l y through the r o o t s . Menge et a l . (1978a) suggests t h a t i t i s the c o n c e n t r a t i o n of P w i t h i n the r o o t and not s o i l P t h a t i s the l i m i t i n g f a c t o r i n m y c o r r h i z a l c o l o n i z a t i o n . T h i s c o u l d e x p l a i n why the VAM f u n g i are e l i m i n a t e d or d r a s t i c a l l y reduced by t h i s f e r t i l i z e r . The f e r t i l i z e r 11-55-0 g i v e s a f l u s h of shoot growth a t the expense of r o o t growth i n p l a n t s grown i n ARD s o i l s . T h i s 100 response of the s e e d l i n g s t o 11-55-0 i n ARD s o i l s should be t r e a t e d w i t h c a u t i o n . In orchards t h a t are b e i n g p l a n t e d t o dwarf t r e e s , w e l l developed r o o t systems are i m p e r a t i v e t o the s u r v i v a l o f the t r e e . I f 11-55-0 causes a r e d u c t i o n i n r o o t growth, these t r e e s may not do w e l l i n orchards i n the l o n g term. Reduced r o o t s are s u s c e p t i b l e t o t o p p l i n g by wind and t o i n v a s i o n of pathogens. While the s h o r t term response of shoot growth t o 11-55-0 i s s t r i k i n g , l o n g term e f f e c t s of 11-55-0 on d i f f e r e n t c l o n a l s t o c k s must be examined. In summary, the f a c t o r s r e s p o n s i b l e f o r ARD may be i n h i b i t i n g the performance of the VAM f u n g i i n nontreated ARD s o i l s . The e x t r a m a t r i c a l hyphae were not examined i n t h i s study, but they may be n e g a t i v e l y a f f e c t e d i n these s o i l s . M i c r o f a u n a l p r e d a t o r s may be f e e d i n g on the e x t r a m a t i c a l hyphae, thereby r e d u c i n g the e f f e c t i v e n e s s of the VAM fungus. Future work i s necessary t o determine the c o r r e l a t i o n between i n t r a - and e x t r a m a t r i c a l hyphae i n apples grown i n ARD s o i l s . The VAM f u n g i may be behaving as pathogens by i n c r e a s i n g the e f f e c t s of indigenous or i n t r o d u c e d f a c u l t a t i v e pathogens. Or they may be e f f e c t i v e l y competing f o r carbohydrates with the h o s t , c a u s i n g a d e l e t e r i o u s C d r a i n , g i v i n g l i t t l e P i n r e t u r n f o r the C r e c e i v e d . The VAM f u n g i are however, i n d u c i n g r o o t growth, which i n the long term e s t a b l i s h e s h e a l t h i e r p l a n t s b e t t e r a b l e t o s u r v i v e t r a n s p l a n t i n g and the i n v a s i o n of r o o t pathogens. While VAM f u n g i do not overcome ARD i n s o i l s used i n t h i s 101 study f u r t h e r long term f i e l d s t u d i e s must be performed with these f u n g i and other ARD s o i l s . An optimal l e v e l o f 11-55-0 f e r t i l i z e r s h o u l d be e s t a b l i s h e d i n which p l a n t growth i n c r e a s e s o c c u r , but a t which no adverse e f f e c t s on the VAM f u n g i o c c u r . 102 CONCLUSIONS The procedures leading to the production of apples are numerous and complicated. In the nursery, c e r t a i n minimum standards must be maintained to ensure healthy rootstock reaches the orchard. Heavy applications of f e r t i l i z e r s , p esticides and i r r i g a t i o n are part of current nursery practices, and lead to the production of healthy rootstock. These practices however, are not conducive to the s u r v i v a l and p r o l i f e r a t i o n of VAM fungi. In the stoolbed nursery used i n t h i s study a l l beds are fumigated p r i o r to establishment. The VAM fungi are severely l i m i t e d i n t h i s nursery as a r e s u l t of t h i s practice. The s o i l i n the budded nursery used i n t h i s study i s never fumigated so the VAM fungi i n th i s nursery are not adversely effected. Within 6 months of fumigation, VAM population should begin to r e e s t a b l i s h and p r o l i f e r a t e . In the stoolbed nursery, t h i s does not appear to be happening. Reestablishment of the fungi appears to be e r r a t i c with minimal spread through the nursery. I t i s hypothesized that c u l t u r a l practices, such as pesticide, f e r t i l i z e r and i r r i g a t i o n applications throughout the growing season, may be responsible for t h i s pattern. The d i f f e r e n c e i n mycorrhizal colonization among the di f f e r e n t rootstocks found i n stoolbed nurseries would r e f l e c t t h i s e r r a t i c pattern of VAM p r o l i f e r a t i o n . This difference i n VAM colonization among the d i f f e r e n t rootstocks i s due primarily to the physical location of each rootstock block i n r e l a t i o n to a viabl e source of inoculum rather than a genotypic receptiveness 103 of host t o fungus. However, r o o t morphology and spread p l a y s an important r o l e i n the meeting of fungus and h o s t . Those r o o t s t o c k s t h a t have the a b i l i t y t o spread throughout the s o i l a rea are more l i k e l y t o encounter a l i v e VAM propagule i n c r e a s i n g t h e i r p r o b a b i l i t y of forming mycorrhizae. M a i l i n g 2 has t h i s c a p a b i l i t y , and t h i s i s most l i k e l y the reason f o r i t s c o n s i s t e n t l y h i g h e r VAM c o l o n i z a t i o n compared t o o t h e r r o o t s t o c k s . Through hormone p r o d u c t i o n , VAM f u n g i may be i n d i r e c t l y i n f l u e n c i n g the r o o t morphology of each c l o n a l r o o t s t o c k . Those r o o t s t o c k s t h a t form hi g h VAM c o l o n i z a t i o n s may have d i f f e r e n t l e v e l s of growth hormones t h a t change t h e i r r o o t morphology. A s e l f - s e r v i n g r e l a t i o n s h i p might e x i s t i n which the fungus c o l o n i z e s the h o s t , changes the r o o t hormone l e v e l s , i n f l u e n c i n g the a b i l i t y of the c l o n e t o s pread, and thereby i n c r e a s i n g the p r o b a b i l i t y of an encounter between r o o t and fungus. The d i f f e r e n c e i n VAM c o l o n i z a t i o n amongst the d i f f e r e n t c l o n e s a f t e r c o l d storage again i s not a g e n o t y p i c f a c t o r , but r a t h e r a r e f l e c t i o n of management p r a c t i c e s . H a r v e s t i n g of r o o t s t o c k s i n s t o o l b e d s i s not random, but occurs by r o o t s t o c k . Each r o o t s t o c k i s t h e r e f o r e processed and s t o r e d as a b l o c k . Those r o o t s t o c k s , t h a t are s t o r e d under i n a p p r o p r i a t e c o n d i t i o n s may have t h e i r VAM a s s o c i a t i o n s a d v e r s e l y a f f e c t e d . M a i l i n g 4 was s t o r e d under dry c o n d i t i o n s , and t h i s may have caused the decrease i n VAM c o l o n i z a t i o n . M a i l i n g Merton 111 was p l a c e d i n c o l d s t o r a g e f i r s t , i n the p a r t of the chamber t h a t r e c e i v e s the 104 l e a s t amount o f oxygen. The l e n g t h o f time i n c o l d s t o r a g e , p l u s a decrease i n the oxygen l e v e l s c o u l d account f o r the decrease i n VAM c o l o n i z a t i o n i n t h i s r o o t s t o c k . E s t a b l i s h i n g p r o d u c t i v e orchards i n a minimal time frame i s an i n t e n s i v e procedure. There are a number of s o i l - b a s e d problems t h a t cause f a i l u r e o f newly t r a n s p l a n t e d apple o r c h a r d s . These problems can be separated by c a u s a l agent. Those a b i o t i c agents, such as s o i l c h e m i c a l , n u t r i e n t and p h y s i c a l imbalances are important though not of d i r e c t concern i n the ARD concept. The ARD concept i s c o n s t a n t l y changing and i s d i f f i c u l t t o e l u c i d a t e . C l e a r l y , a f f e c t e d t r a n s p l a n t s are never k i l l e d , though t h e i r r o o t s are p o o r l y branched and t h e i r shoot growth i s s e v e r e l y i n h i b i t e d . These symptoms are e a s i l y overcome by s o i l f u m i g a t i o n and i n some cases by the a p p l i c a t i o n o f s o i l P as 11-55-0 even though a v a i l a b l e s o i l P i s h i g h . The b i o t i c agents c a u s i n g these symptoms may be separated on the b a s i s o f d i a g n o s i s ; those pathogens t h a t are e a s i l y i d e n t i f i e d and those organisms t h a t are n o t . I f t r a n s p l a n t s are not growing w e l l due to the presence o f a s p e c i f i c organism, t h i s i s not ARD. U n f o r t u n a t e l y t h e r e i s a tendency by many t o s i m p l i f y a complex phenomena. I t i s not a c c e p t a b l e t o use the term ARD as an umbrella term f o r any t r a n s p l a n t i n g d i s o r d e r t h a t i s overcome by s o i l f u m i g a t i o n . Each s o i l i d e n t i f i e d as having ARD i s unique w i t h i t s own c h e m i c a l , p h y s i c a l and b i o l o g i c a l c h a r a c t e r i s t i c s . The word d i s e a s e does not g i v e a good i n d i c a t i o n o f the complexity of the 105 problem and should be r e p l a c e d w i t h d i s o r d e r . I t may be time t o s t a r t appending the term ARD w i t h i t s c a u s a l agent. Thus an apple r e p l a n t problem caused by nematodes should be termed as 'apple r e p l a n t d i s o r d e r caused by nematodes'. I f the c a u s a l agent can not be i d e n t i f i e d then the terminology should simply be 'apple r e p l a n t d i s o r d e r caused by unknown f a c t o r s (ARD-UF)'. A l l 5 s o i l s used i n t h i s study were i d e n t i f i e d as having ARD caused by unknown f a c t o r s . A l l 5 s o i l s had extremely h i g h amounts of phosphorus y e t t e s t s e e d l i n g s grown i n 4 of the 5 s o i l s showed i n c r e a s e s i n shoot growth a f t e r the a d d i t i o n of 11-55-0 and 34-0-0 f e r t i l i z e r s . While g i v i n g an i n c r e a s e i n shoot h e i g h t , the 11-55-0 f e r t i l i z e r reduced r o o t growth and VAM c o l o n i z a t i o n i n t e s t s e e d l i n g s a t the r a t e used. The 34-0-0 f e r t i l i z e r , w h i l e not g i v i n g as g r e a t a shoot response compared t o the 11-55-0, does not appear t o be so severe on VAM a s s o c i a t i o n s . While not g i v i n g a s t r o n g shoot growth i n n o n s t e r i l i z e d s o i l s , the f e r t i l i z e r 0-45-0 r e s u l t s i n i n c r e a s e d r o o t growth and does not a d v e r s e l y a f f e c t VAM c o l o n i z a t i o n . VAM f u n g i appear t o mimic the f e r t i l i z e r 0-45-0 i n ARD s o i l s used i n t h i s s tudy. VAM f u n g i w i l l not overcome ARD. In n o n s t e r i l i z e d s o i l , t h e r e i s l i t t l e shoot growth i n response t o i n o c u l a t e d VAM f u n g i . However, r o o t growth i s i n c r e a s e d by these f u n g i . I t i s apparent t h a t i n o c u l a t e d VAM f u n g i w i l l not overcome ARD i n n o n s t e r i l i z e d s o i l s , and t h a t they do not g i v e as g r e a t a shoot growth response as the 11-55-0 f e r t i l i z e r i n s t e r i l i z e d s o i l s . However, i t i s proposed t h a t with the s t r o n g r o o t based p l a n t s induced by the 106 VAM f u n g i , e v e n t u a l l y these p l a n t s w i l l show s t r o n g shoot growth as w e l l . In n o n s t e r i l i z e d ARD s o i l s , VAM f u n g i are not i n d u c i n g the expected growth of the h o s t . I t i s hypothosized t h a t VAM f u n g i i n n o n - s t e r i l i z e d ARD s o i l s are a c t i n g as pathogens r a t h e r than symbionts. The b e n e f i t s of having t h i s a s s o c i a t i o n i s not mutual. The fungus i s d e r i v i n g i t s carbon source from i t s h o s t , but the P g a i n by the host may have minimal a f f e c t s on apple growth. The fungus i s a b l e t o spread throughout the r o o t , but t h i s i s not an i n d i c a t i o n of spread throughout the s o i l . I t i s f u r t h e r proposed t h a t VAM p r e d a t o r s i n ARD s o i l s are i n d i r e c t l y i n v o l v e d i n ARD. The VAM p r e d a t o r s , may be s i g n i f i c a n t l y r e d u c i n g the e x t r a m a t r i c a l hyphae thereby r e d u c i n g the r o u t e f o r s o i l P movement i n t o the h o s t . They are e l i m i n a t e d upon s t e r i l i z a t i o n , and the fungus i s then a b l e t o perform as expected and m y c o r r h i z a l p l a n t s w i l l show i n c r e a s e d p l a n t growth. The r o l e of s o i l P i n these s o i l s must be e l u c i d a t e d . A l l 5 s o i l s used i n t h i s study had adequate t o h i g h amounts of s o i l P, y e t s e e d l i n g s responded p o s i t i v e l y t o a p p l i c a t i o n s o f N and P f e r t i l i z e r s . The f e r t i l i z e r 11-55-0 was a p p l i e d a t a r a t e of 3,000 kg/ha. T h i s enormous amount of f e r t i l i z e r , w h i l e g i v i n g a growth response i n pot t e s t s , s u r e l y does not r e f l e c t the r e q u i r e d amounts of 11-55-0 i n the f i e l d . I t i s a l s o apparent t h a t methods used t o determine the amounts of a v a i l a b l e P i n the s o i l are not r e f l e c t i n g the P requirements of the apple p l a n t . I t i s t h e r e f o r e proposed t h a t 107 e i t h e r the standards used t o determine P requirements by apple s e e d l i n g s be r e - e v a l u a t e d and changed a c c o r d i n g l y , or t h a t s o i l P should not be used as a c r i t e r i o n t o measure P requirements of young apple s e e d l i n g s . I t may be more a p p r o p r i a t e t o measure r o o t P i n t e s t s e e d l i n g s t o determine the l e v e l s of P r e q u i r e d and a v a i l a b l e t o the p l a n t . Measuring r o o t P would a l s o e l u c i d a t e the r o l e o f VAM f u n g i i n p l a n t s grown i n these s o i l s . In summary, nursery management p r a c t i c e s may be d e t r i m e n t a l t o VAM a s s o c i a t i o n s formed by r o o t s t o c k s . The d i f f e r e n c e i n c l o n a l VAM a s s o c i a t i o n s i s not a g e n o t y p i c r e c e p t i v e n e s s t o VAM f u n g i but r a t h e r due t o environmental f a c t o r s . T h e r e a r e many f a c t o r s r e s p o n s i b l e f o r f a i l u r e i n e s t a b l i s h i n g new apple o r c h a r d s . Fumigation and a d d i t i o n s of N and P f e r t i l i z e r s w i l l r e s u l t i n i n c r e a s e s i n shoot growth i n ARD s o i l s . Root growth and VAM a s s o c i a t i o n s may be a d v e r s e l y a f f e c t e d by these f e r t i l i z e r s . VAM f u n g i w i l l not overcome ARD-UF i n a f f e c t e d s o i l s , though shoot growth i s i n c r e a s e d i f the s o i l i s fumigated f i r s t . These growth responses are not as g r e a t as t h a t induced by 11-55-0 f e r t i l i z e r . In the s h o r t term, the i n v e r s e response occurs i n r o o t growth. As i t i s i n c o r r e c t t o diagnose a s i n g l e f a c t o r as being r e s p o n s i b l e f o r the ARD complex, so i s i t i n c o r r e c t t o search f o r s i n g l e cures f o r t h i s complex phenomena. The ARD complex becomes more co m p l i c a t e d as evidence presented i n t h i s study i n d i c a t e s t h a t VAM f u n g i can behave as pathogens i n these s o i l s r a t h e r than as symbionts. F u r t h e r work 108 i s needed t o e l u c i d a t e the r o l e o f these f u n g i i n t h e s e s o i l s . 109 REFERENCES B a y l i s , G.T.S. 1967. Experiments on the e c o l o g i c a l s i g n i f i c a n c e o f phycomycetous m y c o r r h i z a s . New P h y t o l . 66: 231-243. Benson, N.R. and R.P. Covey J r . 1976. Response of apple s e e d l i n g s t o z i n c f e r t i l i z a t i o n and m y c o r r h i z a l i n o c u l a t i o n . H o r t S c i . 11: 252-253. Benson, N.R. , R.P. Covey J r . , and W. Haglund. 1978. The apple r e p l a n t problem i n Washington S t a t e . J . Amer. Soc. Hort S c i . 103: 156-158. Buszard, D.J. and P. Jensen. 1986. A note on the i n c i d e n c e of apple r e p l a n t d i s e a s e i n Quebec o r c h a r d s . P h y t o p r o t e c t i o n . 67: 133-136. Buwalda, J.G. and K.M. Goh. 1982. Host-fungus c o m p e t i t i o n f o r carbon as a cause of growth d e p r e s s i o n s i n v e s i c u l a r -a r b u s c u l a r m y c o r r h i z a l r y e g r a s s . S o i B i o l . Biochem. 14: 103-105. Cataska, V., V. Vancura, Z. P r i k r y l l and G. Hudska. 1988. A r t i f i c i a l i n d u c t i o n of apple r e p l a n t problem by P e n i c i l l i u m  c l a v i f o r m e i n o c u l a t i o n . P l a n t and S o i l . 107: 127-136. Cataska, V., V. Vancura, G. Hudska and Z. P r i k r y l . 1982. Rhizosphere micro-organisms i n r e l a t i o n t o the apple r e p l a n t problem. P l a n t and S o i l 69: 187-197. Cooper, K. M. 1983. M y c o r r h i z a l f u n g i can improve growth of h o r t i c u l t u r a l c r o p s . O r c h a r d i s t . N.Z. 56: 410-413. Coker, E.G. 1958. Root s t u d i e s : X I I . Root systems of apple on M a i l i n g r o o t s t o c k s on f i v e s o i l s e r i e s . J . Hort S c i . 33: 71-79. Covey, R.P., B.L. Koch and W.A. Haglund. 1984. C o n t r o l of apple r e p l a n t d i s e a s e w i t h formaldehyde i n Washington. P l a n t Disease 68: 981-983. Covey, R.P., B.C. Koch and H.J. L a r s e n . 1981. I n f l u e n c e of v e s i c u l a r - a r b u s c u l a r mycorrhizae on the growth of apple and corn i n low phosphorous s o i l s . Phytopath. 71: 712-715. D e a l , D.R., W.F. Mai and C.W. Boothroyd. 1971. A survey o f b i o t i c r e l a t i o n s h i p s i n grape r e p l a n t s i t u a t i o n s . Phytopath. 62: 503-507. Eno, C. F. and H. Popenoe. 1963. The e f f e c t of gamma r a d i a t i o n on the a v a i l a b i l i t y of n i t r o g e n and phosphorus i n s o i l . S o i l 110 S c i . Soc. Amer. Proc. 28: 299-301. G e n n a r i , M., M. Negre and R. A m b r o s o l i . 1987. E f f e c t s of ethylene oxide on s o i l m i c r o b i a l content and some s o i l chemical c h a r a c t e r i s t i c s . P l a n t and S o i l . 102: 197-200. G i o v a n n e t t i , M. and B. Mosse. 1980. An e v a l u a t i o n of technigues f o r measuring v e s i c u l a r - a r b u s c u l a r m y c o r r h i z a l i n f e c t i o n i n r o o t s . New P h y t o l . 84: 489-500. Granger, R..L., C. P l e n c h e t t e and J.A. F o r t i n . 1983. E f f e c t of a v e s i c u l a r - a r b u s c u l a r (VA) m y c o r r h i z a l f u n g u s (Glomus  epigaeum) on the growth and l e a f m i n e r a l content of two apple c l o n e s propagated i n v i t r o . Can. J . P l a n t S c i . 63: 551-555. Grochowska, M. , G.J. Buta, G.L. S t e f f e n s and M. F a u s t . 1984. Endogenous auxin and g i b b e r e l l i n l e v e l s i n low and h i g h v i g o u r apple s e e d l i n g s . In: Acta H o r t . 146: I n t e r n a t i o n a l Workshop on C o n t r o l l i n g V i g o r i n F r u i t T r e e s . Ed: F a u s t , M. I n t . Soc. H o r t . Sci.Wageningen, N e t h e r l a n d s , pp 125-134. H a l l , I.R. and A. K e l s o n . 1981. An improved technque f o r the p r o d u c t i o n of endomycorrhizal i n f e s t e d s o i l p e l l e t s . N.Z. J . A g r i c . 24:221-222. Hartman, H.T. and D.E. K e s t e r . 1975. P l a n t Propagation: P r i n c i p l e s and P r a c t i c e s . P r e n t i c e - H a l l I n c . , Englewood C l i f f s , New J e r s e y , pp.465. H a t t i n g h , M.J., and J.W. Gerdemann. 1975. I n o c u l a t i o n of b r a z i l i a n sour orange seed w i t h an endomycorrhizal fungus. Phytopath. 65: 1013-1016. H a v i s , I . , M o r r i s H.F., R. Manning and T.E. Denman. 1958. Responses of r e p l a n t e d peach t r e e s t o s o i l treatments i n f i e l d t e s t s i n Texas. P r o c . Amer. Soc. H o r t . S c i . 71: 67-76. Hoepfner, E.F., G.L. Koch and R.P. Covey. 1983. Enhancement of growth and phosphorus c o n c e n t r a t i o n i n apple s e e d l i n g s by v e s i c u l a r - a r b u s c u l a r mycorrhizae. J . Amer. Soc. H o r t . S c i . 108: 207-209. H o e s t r a , H. 1968. Replant d i s e a s e s of apple i n the N e t h e r l a n d s . Meded. Landbouw. Wagen., Nederland. Veenman and Zonen Wageningen. H o e s t r a , H. and Oostenbrink. 1962. Nematodes i n r e l a t i o n t o p l a n t growth. IV. P r a t y l e n c h u s penetrans (Cobb) on o r c h a r d t r e e s . Neth. J . A g r i c . S c i . 10: 286-296. Hwang, S. F. 1988. E f f e c t s of v e s i c u l a r a r b u s c u l a r mycorrhizae 111 and m e t a l a x y l on growth of a l f a l f a s e e d l i n g s i n s o i l s from f i e l d s w i t h " A l f a l f a S i c k n e s s " i n A l b e r t a . P l a n t D i s . 72: 448-452. J a f f e , B.A., G.S. Abawi and W.F. Mai. 1982. Role of s o i l m i c r o f l o r a and Pra t y l e n c h u s penetrans i n an apple r e p l a n t d i s e a s e . Phytopath. 72: 247-251. K e l l y , C C . and R.H. S p r i l s b u r y . 1949. S o i l Survey of the Okanagan and Similkameen V a l l e y s : B r i t i s h Columbia. B.C. Dept. A g r i c . Rep. 3. V i c t o r i a , B.C. K l e i n s c h m i d t , G.D. and J.W. Gerdemann. 1972. S t u n t i n g of c i t r u s s e e d l i n g s i n fumigated nursery s o i l s r e l a t e d t o the absence of endomycorrhizae. Phytopath. 62: 1447-1453. Kormanik, P.P. and A.C. McGraw. 1982. Q u a n t i f i c a t i o n of v e s i c u l a r - a r b u s c u l a r mycorrhizae i n p l a n t r o o t s . I n . Methods and P r i n c i p l e s of M y c o r r h i z a l Research. Ed. N.C. Schenck. APS. S t . P a u l , Minnesota, pp 37-46. La Rue, J.H., W.D. M c C l e l l a n and W.L. Peacock. 1975. M y c o r r h i z a l f u n g i and peach nursery n u t r i t i o n . C a l . A g r i c . 29: 6 - 7 . L a v k u l i c h , L.M. 1978. Phosphorus s o l u b l e i n Sodium Carbonate. In Methods Manual: Pedology L a b o r a t o r y . Dept S o i l S c i e n c e . U n i v . B.C. Vancouver, B.C. L i e g e l L.H. 1983. E f f e c t o f dry-heat s t e r i l i z a t i o n on chemical p r o p e r t i e s o f Puerto Rican s o i l s . Commun. S o i l S c i . P l a n t A n a l . 14: 277-286. L u t t e r m e r d i n g , H.A. 1981. S o i l s o f the Langley Vancouver Map Area: Report 15. v o l (6) B.C. Min. E n v i r o n . , V i c t o r i a , B.C. Lu t t e r m e r d i n g , H. A. 1980. S o i l s of the Langley-Vancouver Map Ar e a , b u l l 18.vol (2) B.C. Min. E n v i r o n . V i c t o r i a , B.C. Mack, A.R. 1963. B i o l o g i c a l a c t i v i t y and m i n e r a l i z a t i o n of n i t r o g e n i n t r e e s o i l s as induced by f r e e z i n g and d r y i n g . Can. J . S o i l S c i . 43: 316-324. Mai, W.F. and G.S. Abawi. 1981. C o n t r o l l i n g r e p l a n t d i s e a s e s of pome and stone f r u i t s i n n o r t h e a s t e r n U n i t e d S t a t e s by p r e p l a n t f u m i g a t i o n . P l a n t D i s . 65: 859-863. Menge, J.A. 1982. E f f e c t of s o i l fumigants and f u n g i c i d e s on v e s i c u l a r - a r b u s c u l a r f u n g i . Phytopath. 72: 1125-1132. Menge, J.A., D. S t i e r l e , D.J. Ba g y a r a j , E.L.V. Johnson and R.T. L e o n a r d . 1978a. Phosphorus c o n c e n t r a t i o n i n p l a n t s r e s p o n s i b l e f o r i n h i b i t i o n of m y c o r r h i z a l i n f e c t i o n . New. 112 P h y t o l . 80: 575-578. Menge, J.A., R.M. D a v i s , E.L.V. Johnson and G.A. Zentmyer. 1978b. M y c o r r h i z a l f u n g i i n c r e a s e growth and reduce t r a n s p l a n t i n j u r y i n avocado. C a l . A g r i c . 32: 6-7. M i l l e r , D.D., P.A. Domoto and C. Walker. 1985a. M y c o r r h i z a l f u n g i a t e i g h t e e n apple r o o t s t o c k p l a n t i n g s i n the U n i t e d S t a t e s . New P h y t o l . 100: 379-391. M i l l e r , D.D., P.A. Domoto and C. Walker. 1985b. C o l o n i z a t i o n and e f f i c a c y of d i f f e r e n t endomycorrhizal f u n g i w i t h apple s e e d l i n g s a t two phosphorus l e v e l s . New. P h y t o l . 100: 393-402. Mosse, B. 1957. Growth and chemical composition of m y c o r r h i z a l and non-mycorrhizal a p p l e s . Nature 179: 922-924. Mountain, W.B. and H.R. Boyce. 1958. The peach r e p l a n t problem i n O n t a r i o . V I . The r e l a t i o n s h i p of P r a t y l e n c h u s penetrans t o the growth of young peach t r e e s . Can. J . Bot. 36: 135-151. Nemec, S. 1987. E f f e c t of storage temperature and moisture on Glomus s p e c i e s and t h e i r subsequent e f f e c t on c i t r u s r o o t s t o c k s e e d l i n g growth and mycorrhiza development. Trans. B r i t . M ycol. Soc. 89: 205-212. Pe p i n , H.S., W.F. S e w e l l , and J . F . W i l s o n . 1975. S o i l p o p u l a t i o n s of T h i e l a v i o p s i s b a s i c o l a a s s o c i a t e d w i t h c h e r r y r o o t s t o c k i n r e l a t i o n t o e f f e c t s of the pathogen on t h e i r growth. Ann. A p p l . B i o l . 79: 171-176. P l e n c h e t t e , C. , V. F u r l a n and J.A. F o r t i n . 1982. E f f e c t s of d i f f e r e n t endomycorrhizal f u n g i on f i v e host p l a n t s grown on c a l c i n e d m o n t m o r i l l o n i t e . J . Amer. Soc. H o r t . S c i . 107: 535-538. R e i c h , L. 1988. Rates of i n f e c t i o n and e f f e c t s of 5 v e s i c u l a r -a r b u s c u l a r m y c o r r h i z a l f u n g i on a p p l e s . Can. J . P l a n t S c i . 68: 233-239. R e i d , C P . P. and G.D. Bowen. 1979. E f f e c t s of s o i l moisture on V/A mycorrhiza formation and r o o t development i n Medicago. In: The S o i l - R o o t I n t e r f a c e . Ed. H a r l e y , J . L . and R.S. R u s s e l l . Academic P r e s s , London, pp 211-220. Robertson, W.J., C D . Boyle and H.L. Brown. 1988. Endomycorrhizal s t a t u s of c e r t i f i e d strawberry nursery s t o c k . J . Amer. Soc. H o r t . S c i . 113: 525-529. Rom, R.C. and S.A. Brown. 1973. Burr-knot c h a r a c t e r i s t i c s of s i x c l o n a l r o o t s t o c k s . F r u i t V a r. J . 27: 84-86. 113 Rom, R.C. and S.A. Brown. 1979. F a c t o r s a f f e c t i n g burrknot f o r m a t i o n on c l o n a l Malus r o o t s t o c k . H o r t S c i . 14: 231-232. Rom, R.C. and G.R. M o t i c h e k . 1987. C u l t i v a r e f f e c t on a d v e n t i t i o u s r o o t development o f c l o n a l apple r o o t s t o c k s . H o r t S c i . 22: 57-58. Ross, R.G., K.B. McRae, and R.J. Newberry. 1984. Growth o f apple t r e e s a f f e c t e d by r e p l a n t d i s e a s e a f t e r l i f t i n g , s i t e f u m i g a t i o n , and r e p l a n t i n g . Can. J . P l a n t Path. 6: 257-258. Ryan, C.L.J. 1975. S p e c i f i c r e p l a n t d i s e a s e s i n Hawkes Bay. P a r t I I . Pot t e s t s used f o r d e t e c t i n g r e p l a n t d i s e a s e . I t s i d e n t i f i c a t i o n and d i s t r i b u t i o n . O r c h a r d i s t N.Z. 48: 165-168. S a l o n i u s , P.O, J.B. Robinson, and F.E. Chase. 1967. A comparison of a u t o c l a v e d and gamma-irradiated s o i l s as media f o r m i c r o b i a l c o l o n i z a t i o n experiments. P l a n t and S o i l . 2: 239-249. Savory, B.M. 1967. S p e c i f i c r e p l a n t d i s e a s e s o f apple and c h e r r y . Rep. E. M a i l i n g Res. S t n . f o r 1966. pp. 205-208. S e w e l l , G.W.F. 1981. E f f e c t s of Pythium s p e c i e s on the growth of apple and t h e i r p o s s i b l e c a u s a l r o l e i n apple r e p l a n t d i s e a s e . Ann. A p p l . B i o l . 97: 31-42. S e w e l l , G.W.F. 1984. Replant d i s e a s e : Nature, e t i o l o g y and importance. B r i t . Crop P r o t e c t . Conf.: Pests and D i s . 3: 1175-1182 S e w e l l , G.W.F. and A.L. Roberts. 1984. E f f e c t s o f phosphorus and VA mycorrhiza on apple growth. Rep. East M a l l . Res. S t a . 1983 pp 101-102. S e w e l l , G.W.F. and A.L. Roberts. 1985. Small responses t o s o i l s t e r i l i s a t i o n i n apple s e e d l i n g b i o - a s s a y s of orc h a r d s o i l s . Rep. East M a l l . Res. S t a . 1985 pp. 113-115. S e w e l l , G.W.F. and G.C. White. 1979. The e f f e c t s o f f o r m a l i n and oth e r s o i l treatments on the r e p l a n t d i s e a s e o f a p p l e . J . H o r t . S c i . 54: 333-335. S e w e l l , G.W.F. and G.C. White. 1981. E f f e c t s o f f o r m a l i n and oth e r s o i l treatments on the poor growth o r r e p l a n t d i s e a s e of a p p l e . Rep. E. M a l l . Res. S t n . f o r 1980. pp 169-170. S k i p p e r , H.D. and D.T. Westermann. 197 3. Comparative e f f e c t s of propylene o x i d e , sodium a z i d e and a u t o c l a v i n g on s e l e c t e d s o i l p r o p e r t i e s . S o i l B i o l . Biochem. 5: 409-414. 114 S l y k h u i s , J .T. and T.S.C. L i . 1985. Responses of apple s e e d l i n g s t o b i o c i d e s and phosphate f e r t i l i z e r s i n o r c h a r d s o i l s i n B r i t i s h Columbia. Can. J . P l a n t Path. 7: 294-301. T r a q u a i r , J . A. and S.M Berch. 1988. C o l o n i z a t i o n o f peach r o o t s t o c k by indigenous v e s i c u l a r - a r b u s c u l a r m y c o r r h i z a l (VAM) f u n g i . Can. J . P l a n t S c i . 68: 893-898. T r u d g i l l , D.L. 1984. A pot i n v e s t i g a t i o n i n t o the nature o f r a s p b e r r y (Rubus i d a e n s L.) r e p l a n t i n g d i s o r d e r s i n S c o t l a n d . Crop Res. (Hort. Res.) 24: 105-118. Upstone, M.E. 1974. The t e s t i n g of orchard s o i l s f o r s p e c i f i c apple r e p l a n t d i s e a s e . 1965-1974. ADAS. S c i . S e r . Ann. Rep: pp 181-185. Utkhede, R. 1987. New l i n e s of r e s e a r c h on apple r e p l a n t d i s e a s e . In . S u c c e s s f u l R e p l a n t i n g . Ed. McPhee, B. and T. Swales. Okan. S i m i l . Coop. Grow. Assoc. pp 32-34. Warnock, A . J . , F i t t e r , A.H. and M.B. Usher. 1982. The i n f l u e n c e of a s p r i n g t a i l Folsomia Candida ( I n s e c t , Collembola) on the m y c o r r h i z a l a s s o c i a t i o n of l e e k A l l i u m porrum and the v e s i c u l a r - a r b u s c u l a r m y c o r r h i z a l e n d o p h y t e Glomus  f a s c i c u l a t u s . New. P h y t o l . 90:285-292. Warcup, J.H. 1957. Chemical and b i o l o g i c a l a s p e c t s o f s o i l s t e r i l i z a t i o n . S o i l s and F e r t i l i z e r s . 20: 1-5. W i l l i a m s - L i n e r a , G. , and J . J . Ewel. 1984. E f f e c t o f a u t o c l a v e s t e r i l i z a t i o n o f t r o p i c a l andept on seed germ i n a t i o n and s e e d l i n g growth. P l a n t and S o i l . 82: 263-268. Zar, J.H. 1984. B i o s t a t i s t i c a l a n a l y s i s . P r e n t i c e H a l l . Englewood C l i f f s . N.J. pp. 239-240. 115 APPENDIX S o i l pH f o r Old Rows and Alleyways S o i l s 1 2 3 Mean 7.40 7.34 7.39 7.37 7.13 7.09 7.10 7.11 7.41 7.20 7.44 7.38 7.50 7.41 7.43 7.43 7.43 7.47 7.47 7.45 7.06 7.11 7.08 7.09 7.23 7.27 7.26 7.25 7.32 7.33 7.28 7.31 7.36 7.28 7.24 7.29 6.83 6.89 6.90 6.86 7.42 7.43 7.43 7.43 7.30 7.36 7.35 7.34 MEAN 7.27 Table A . l a : S o i l pH f o r Old Rows: S o i l 1 1 2 3 Mean 7.41 7.51 7 . 48 7.47 7.49 7.51 7. 48 7.49 7.47 7.43 7. 54 7.49 7 .27 7.20 7. 28 7.25 6. 62 6. 68 6.64 7.40 7.28 7. 35 7.34 7.70 7.09 7. 19 7.33 7.21 7.16 7. 19 7.19 7.30 7.19 7. 28 7.26 7.05 7.00 7. 06 7.04 7.10 7 .11 7. 10 7 .10 7.39 7.30 7. 35 7.34 6. 60 6.49 6. 52 6. 56 7.20 7.17 7. 13 7.20 MEAN 7.19 Table A . l b : S o i l pH f o r Old Alleyways: S o i l 1 116 1 2 3 Mean 7.32 7.42 7.33 7.36 7.50 7.39 7.30 7.40 6.99 6.54 7.32 6.94 7.60 7.59 7.59 7.60 7.00 7.23 7.27 7.16 7 .39 7 .48 7.34 7 .40 7.25 7.33 7.32 7.30 7.44 7.40 7.42 7.42 7.00 7.02 6.99 7.02 7.40 7.41 7.45 7.42 7.07 7.11 7.12 7.10 7.34 7.28 7.17 7.26 7.18 7.14 7.14 7.15 7.28 7.29 7.35 7.30 7. 61 7.50 7.54 7.55 MEAN 7.29 Table A.2a: S o i l pH f o r Old Rows: S o i l 2 1 2 3 Mean 7.57 7.51 7.49 7.52 7.32 7.30 7.33 7.32 7.54 7.55 7.53 7.54 7.31 7.33 7.34 7.34 7.58 7.63 7.60 7. 60 7.45 7.49 7.54 7.49 7.29 7.23 7 . 39 7.30 7.19 7.08 6.98 7.08 7.53 7 . 50 7.49 7.51 7.59 7.54 7.55 7.55 7.74 7.74 7.81 7.76 7.44 7.49 7.48 7.47 '7.45 7.48 7.40 7.44 MEAN 7.46 Table A.2b: S o i l pH f o r Old Alleyways: S o i l 2 117 1 2 3 Mean 5.90 5.81 6.06 5.92 6.86 7.26 6.92 7.01 5.19 5.67 5.88 5.58 6.95 7.04 6.99 6.99 6.46 6.19 6.31 6.32 5.73 5.63 5.64 5.66 6.11 6.14 6.13 6.12 5. 63 5. 64 5.68 5.65 6.41 6.30 6.31 6.34 6.45 6.25 6.25 6.31 5.99 5.61 5.78 5.79 7.27 7.08 7.12 7.15 7.65 7.54 7.64 7.61 MEAN 6.34 Table A.3a: S o i l pH f o r Old Rows: S o i l 3 1 2 3 Mean 6. 15 6.20 6.18 6.17 6.55 6.77 6.63 6. 65 6.91 6.99 6.98 6.96 7.30 7.41 7.50 7.40 5.78 5.55 5.64 5.65 6.78 7.06 7.34 7.06 5.54 5.42 5.47 5.47 6. 05 5.94 6. 01 6. 00 5.84 5.78 5.80 5.80 5.76 5.76 5.83 5.78 7.21 7.10 7.09 7.13 7 . 00 6.80 6.83 6. 87 5.94 5.78 5.77 5.83 MEAN 6.37 Table A.3b: S o i l pH f o r Old Alleyways: S o i l 3 118 1 2 3 Mean 7.71 7.71 7. 69 7.70 7.10 7.14 7 .13 7.12 7.45 7.37 7.44 7.42 7.65 7.36 7.36 7.45 7.71 7.56 7.64 7. 63 7.65 7.72 7.66 7.67 6.96 6.94 6.96 6.95 7.53 7.39 7.39 7.43 7.54 7.51 7.56 7.53 7.06 7.02 7.04 7.04 7.55 7.52 7.51 7.52 6.83 6.79 6.71 6.77 7.39 7.62 7.31 7.44 7.78 7.77 7.74 7.76 MEAN 7.39 Ta b l e A.4a: S o i l pH f o r Old Rows: S o i l 4 1 2 3 Mean 6.95 6.97 7.03 6.98 7.61 7.57 7. 60 7.59 7.29 7.21 7.26 7.25 7.48 7.38 7.35 7.40 7.15 7.05 7.04 7. 08 7.43 7.39 7.36 7.39 7.52 7 . 49 7.56 7.52 7.21 7.15 7.13 7.16 6.96 6.89 6.81 6.88 7.35 7 .33 7.14 7.27 7.14 7.13 7.13 7 .13 7.40 7 .23 7.26 7.29 7.05 7.97 7.01 7.04 7. 04 6.98 7.02 7.01 MEAN 7.21 Ta b l e A.4b: S o i l pH f o r Old Alleyways: S o i l 4 119 Shoot Height For S o i l s 1 - 4 wit h V a r i o u s F e r t i l i z e r and S t e r i l i z a t i o n Treatments Non S t e r i l e S t e r i l e C o n t r o l 8.25 a* 14.13 a,b 11-55-0 17.00 b,c 21.88 b,c 34-0-0 14.50 a,b,c 23.67 c 0-45-0 8.33 a 17.50 b,c able A.5: Mean Shoot Height (cm): S o i l 1 Non S t e r i l e S t e r i l e C o n t r o l 8.53 a,b 13.54 a,b,c 11-55-0 15.83 b,c 19.67 c 34-0-0 14.00 a,b,c 18.08 c 0-45-0 7.08 a 14.45 a,b,c Table A.6: Mean Shoot Height (cm): S o i l 2 Non S t e r i l e S t e r i l e C o n t r o l 11-55-0 34-0-0 0-45-0 20.28 a,b* 32.83 b 29.58 b 12.75 a 21.00 a,b 30.08 b 31.83 b 25.75 b Table A.7: Mean Shoot Height (cm): S o i l 3 Non S t e r i l e S t e r i l e C o n t r o l 11-55-0 34-0-0 0-45-0 8.53 a* 15.83 a 14.00 a 7.08 a 13.54 a 19.67 a 18.08 a 14.45 a Table A.8: Mean Shoot Height (cm): S o i l 4 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) 120 Root Dry Weight f o r S o i l 1 and 2 w i t h V a r i o u s F e r t i l i z e r and S t e r i l i z a t i o n Treatments Non S t e r i l e S t e r i l e C o n t r o l 11-55-0 34-0-0 0-45-0 0.160 a* 0.200 a 0.190 a 0.413 a 0.230 a 0.268 a 0.217 a 0.326 a Table A.9: Mean Root Dry Weight (gm): S o i l 1 Non S t e r i l e S t e r i l e C o n t r o l 11-55-0 34-0-0 0-45-0 0.218 a* 0.315 a 0.227 a 0.413 a 0.356 a 0.223 a 0.367 a 0.400 a Table A.10: Mean Root Dry Weight(gm): S o i l 2 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) 121 M y c o r r h i z a l C o l o n i z a t i o n i n S o i l 1 and 2 A f t e r V a r i o u s F e r t i l i z e r and S t e r i l i z a t i o n Treatments Non S t e r i l e S t e r i l e C o n t r o l 11-55-0 34-0-0 0-45-0 42.60 b* 5.66 a 32.25 b 33.78 b 0. 00 a 0.00 a 0.00 a 0. 00 a Table A.11: Percent M y c o r r h i z a l C o l o n i z a t i o n : S o i l 1 Non S t e r i l e S t e r i l e C o n t r o l 11-55-0 34-0-0 0-45-0 52.53 c* 0.55 a 33.17 b 37.95 b 0. 00 a 0.00 a 0. 00 a 0. 00 a Table A.12: Percent M y c o r r h i z a l C o l o n i z a t i o n : * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e Tukey's (HSD) t e s t ) S o i l 2 (a = 0.05 122 Shoot Height A f t e r S o i l S t e r i l i z a t i o n and Adding VAM Fungi and F e r t i l i z e r Non S t e r i l e S t e r i l e GC 10.8 a* 17.0 a,b GV 10.5 a 14.2 a GI 15.3 a,b 13.3 a 11-55-0 22.5 b,c 27.1 c CT 11.6 a 16.4 a,b Table A.13: Mean Shoot Height (cm): S o i l 2 Non P a s t e u r i z e d P a s t e u r i z e d GC 10.1 a,b 12.2 a,b,c GV 10.9 a,b,c 21.3 d GI 10.1 a,b,c 16.8 c,d OR 8.0 a 16.1 b,c,d 11-55-0 7.7 a 30.8 e CT 9.1 a 16.2 b,c,d Table A.14: Mean Shoot Height (cm): S o i l 5 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) 123 Shoot Dry Weight A f t e r S o i l S t e r i l i z a t i o n and Adding VAM Fungi and F e r t i l i z e r Non S t e r i l e S t e r i l e GC 0.403 a* 1.137 b GV 0.275 a 1.106 b GI 0.688 a,b 0.763 a,b 11-55-0 2.098 c 2.153 c CT 0.574 a,b 1.140 b Table A. 15: Mean Shoot Dry Weight (gra) : S o i l 2 Non P a s t e u r i z e d P a s t e u r i z e d GC 0.619 a* 1.220 b,c,d GV 0.817 a,b,c 2.068 e,f GI 0.718 a,b 1.500 d,e OR 0.542 a 1.295 b,c,d 11-55-0 0.355 a 2.467 f CT 0.603 a 1.366 c,d Table A.16: Mean Shoot Dry Weight (gm): S o i l 5 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) 124 Root Dry Weight A f t e r S o i l S t e r i l i z a t i o n and Adding VAM Fungi and F e r t i l i z e r Non S t e r i l e S t e r i l e GC 0.113 a,b,c* 0.240 b,c,d GV 0.104 a,b 0.332 d GI 0.082 a 0.235 b,c,d 11-55-0 0.241 b,c,d 0.157 a,b,c CT 0.137 a,b,c 0.259 c,d Table A.17: Mean Root Weight (gm): S o i l 2 Non P a s t e u r i z e d P a s t e u r i z e d GC 0.064 a,b* 0.200 b,c,d GV 0.053 a,b 0.303 d GI 0.048 a,b 0.299 d OR 0.035 a 0.260 c,d 11-55-0 0.022 a 0.132 a,b,c CT 0.051 a,b 0.123 a,b,c Table A.18: Mean Root Dry Weight (gm): S o i l 5 * same l e t t e r i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e (a = 0.05 Tukey's (HSD) t e s t ) 125 M y c o r r h i z a l C o l o n i z a t i o n A f t e r S o i l S t e r i l i z a t i o n and Adding VAM Fungi and F e r t i l i z e r Non S t e r i l e S t e r i l e GC 46.70 b , c * 48.93 b , c GV 48.20 b , c 68.03 c GI 60.07 c 66.68 C 11-55-0 13.38 a , b 0.00 a CT 59.35 c 0. 00 a T a b l e A.19: Percent M y c o r r h i z a l C o l o n i z a t i o n : S o i l 2 Non P a s t e u r i z e d P a s t e u r i z e d GC 32.68 b,c 46.10 b,c,d GV 21.80 a,b,c 38.47 b,c,d GI 18.32 a,b 61.52 d OR 0.55 a 48.13 c,d 11-55-0 0. 00 a 0.00 a CT 3.00a 0. 00 a Table A.20: Percent M y c o r r h i z a l C o l o n i z a t i o n : S o i l 5 126 

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