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Vesicular-arbuscular mycorrhizal fungi in winter wheat in South Coastal British Columbia Cade, Barbara Jean 1989

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VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGI IN WINTER WHEAT IN SOUTH COASTAL BRITISH COLUMBIA By BARBARA JEAN CADE B. Sc. (Hons.), Queen's University, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of S o i l Science) We accept t h i s thesis as conforming , to the required standard THE UNIVERSITY OF BRITISH A p r i l 1989 (g) Barbara Jean Cade, COLUMBIA 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 of Sn-il s H p n r p The University of British Columbia Vancouver, Canada . 21 A 'pr- i l 1989 Date DE-6 (2/88) ABSTRACT This two-part study was conducted to examine the VA mycorrhizae of winter wheat i n South Coastal B r i t i s h Columbia. In the f i r s t part, colonization patterns at four s i t e s were monitored through the October to August growing season, as were a variety of environmental fac t o r s which might influence these patterns. F a l l c o lonization was observed at two s i t e s , which were the most highly colonized at a l l f i v e harvests. The other two s i t e s were not colonized u n t i l l a t e spring, and never attained high l e v e l s of i n f e c t i o n . S o i l phosphorus, s o i l moisture and plant physiology were shown to exert the strongest influences on these patterns, while temperature was not believed to be an important factor. In the second part of t h i s study, the systemic fungicide Bayleton (triademifon) was shown to be mildly f u n g i s t a t i c when i t was sprayed onto the f o l i a g e of young plants, and to increase the l e v e l s of some f o l i a r elements. The systemic fungicide T i l t (propiconazole) had no e f f e c t on colonization or f o l i a r nutrients when i t was f o l i a r l y applied to older plants. From t h i s study, i t i s d i f f i c u l t to determine the importance of VA mycorrhizae to wheat i n t h i s region. With the more than adequate l e v e l s of plant nutrients i n the s o i l s at these s i t e s , the mycorrhizae may not be very important n u t r i t i o n a l l y to the host plants. However, they may provide p o s i t i v e e f f e c t s on growth which were not apparent from t h i s study. TABLE OF CONTENTS l o g i c Page Abstract i i Table of Contents i i i L i s t of Tables i v L i s t of Figures v i Acknowledgements v i i i Chapter One: General Introduction 1 Chapter Two: Patterns of Colonization Introduction 5 Materials and Methods 9 Results 23 Discussion 46 Conclusions 54 Chapter Three: E f f e c t s of Fungicides Introduction 55 Materials and Methods 59 Results 63 Discussion 72 Conclusions 77 Chapter Four: General Summary and Conclusions General Summary 78 General Conclusions 82 Li t e r a t u r e Cited , 83 Appendix I: Results of Patterns Study 91 Appendix I I : Fungal C o l l e c t i o n Numbers 94 Appendix I I I : Results of Fungicide Study 95 i i i LIST OF TABLES Table Togic Page I Description of the f i e l d s i t e s 10 II Crop Treatments 13 III Dates of planting and harvests 13 IV Mean monthly a i r temp, and precip. 24 V Normal s o i l temperatures 24 Via Analysis of variance for colonization 26 . b Mean colonization at each s i t e 26 VII Fungal species from each s i t e 31 V i l l a Analysis of variance f o r Bray PI s o i l P 34 b Mean s o i l phosphorus (Bray PI) 34 IXa Analysis of variance f o r Mehlich 3 s o i l P 35 b Mean s o i l phosphorus (Mehlich 3) 35 Xa Analysis of variance f o r s o i l zinc 37 b Mean s o i l zinc at each s i t e 37 Xla Analysis of variance for s o i l copper 38 b Mean s o i l copper at each s i t e 38 X l l a Analysis of variance f o r s o i l pH 39 b Mean s o i l pH at each s i t e 39 X l l l a Analysis of variance f o r f o l i a r P 41 b Mean f o l i a r phosphorus at each s i t e 41 XlVa Analysis of variance for f o l i a r zinc 42 b Mean f o l i a r zinc at each s i t e 42 XVa Analysis of variance f o r f o l i a r copper 43 b Mean f o l i a r copper at each s i t e 43 XVI Correlation matrix for Chapter Two 45 XVII Sampling dates for fungicide t r i a l s 61 XVIIIa Analysis of variance f o r B. colonization 64 b Mean colonization, Bayleton t r i a l 64 XlXa Analysis of variance for B. f o l i a r P 65 b Mean f o l i a r P, Bayleton t r i a l 65 XXa Analysis of variance f o r B. f o l i a r z i nc 66 b Mean f o l i a r zinc, Bayleton t r i a l 66 iv LIST OF TABLES^ CONTINUED Table ZQfiic Page XXIa b Analysis of variance f o r T. colonization Mean colonization, T i l t t r i a l 68 68 XXI l a b Analysis of Mean f o l i a r variance for T. f o l i a r P P, T i l t t r i a l 69 69 XXIIIa b Analysis of Mean f o l i a r variance for T. f o l i a r z inc zinc, T i l t t r i a l 70 70 XXIV Correlation matrix for Chapter Three 71 L i s t of Figures Figure Togic Page 1 Map of the f i e l d s i t e s 10 2 Photographs of the f i e l d s i t e s 11 3 Photographs of the plants at the harvest 15,16 4 Photograph of the Delta s i t e , Feb. harvest 17 5 Photographs of the sampling methods 18 6 Colonization counting method 20 7 Colonization per s i t e through time 26 8 Colonization through thermal time 27 9 Photographs of colonized roots 28 10 Photographs of coarse and f i n e endophyte 29 11 Photographs of the fungal spores 32,33 12 S o i l P (Bray PI) per s i t e through time 34 13 S o i l P (Mehlich 3) per s i t e through time 35 14 S o i l zinc per s i t e through time 36 15 S o i l copper per s i t e through time 38 16 S o i l pH per s i t e through time 39 17 F o l i a r phosphorus per s i t e through time 41 18 F o l i a r z inc per s i t e through time 42 19 F o l i a r copper per s i t e through time 43 20 Photograph of fungicide study area 60 21 Photographs of fungicide t r i a l plants 62 22 E f f e c t of Bayleton on colonization 64 23 E f f e c t of Bayleton on f o l i a r phosphorus 65 24 E f f e c t of Bayleton on f o l i a r zinc 66 vi L i s t of Figures Figure Togic Page 25 E f f e c t of T i l t on colonization 68 26 E f f e c t of T i l t on f o l i a r phosphorus , 69 27 E f f e c t of T i l t on f o l i a r zinc 70 v i i ACKNOWLEDGEMENTS My sincerest thanks go to a l l of the many people who have assisted me i n t h i s project. These include: Dr. Shannon Berch for her. support over the past few years; Drs. Art Bomke and Wayne Temple for allowing me to j o i n t h e i r winter wheat team; ARDSA for funding the o v e r a l l project and the B. C. A g r i c u l t u r a l Services Coordinating Committee for funding my small part i n i t ; NSERC f o r an operating grant to Dr. S. M Berch; Niels Holbek and the s t a f f at the UBC Oyster River Research Farm; Stan Freyman and the s t a f f at the Agriculture Canada Research Station, Agassiz; Hugh and Stan Reynolds at Reynelda Farms; Art and Stewart Frank i n Chilllwack; to Karln S h u l t i s for sampling assistance; a l l of the members of Hut 0-21 - Aaron, B i l l , Elisabeth, Peni, Rola, Shaobing, Sharmin, Tim and Xlnghua - for assistance i n many ways; Ev Wolterson for technical assistance; my committee members - Drs. Ballard, Berch, Bomke and Lavkulich - for t h e i r patience; Drs. Eaton, Kozak and Greig for t h e i r s t a t i s t i c a l advice; and Chuck Menun for h i s many hours of computer work. F i n a l l y , s p e c i a l thanks are extended to my parents, Jim and Marilyn, and to my s i s t e r Linda, for a l l of t h e i r love and support, and for putting up*with me through a l l of t h i s . v i i i CHAPTER ONE GENERAL INTRODUCTION The term "mycorrhiza" was f i r s t introduced by Frank i n 1885 and l i t e r a l l y means "fungus-root". It i s the mutualistic association between a higher plant and a fungal symbiont <Harley and Smith 1983). Vesicular-arbuscular or VA mycorrhizae are the most common of a l l mycorrhizae, and are found i n Pteridophytes, and nearly a l l of the fa m i l i e s of the Gymnosperms and Angiosperms. The fungi forming these associations are found i n s o i l s throughout the world, and show l i t t l e host s p e c i f i c i t y . They belong to the genera Glomus, Gigasggra, AoaulgsQgra, Entroghoepora, §outellosp.ora, and Sclergcy_stis i n the Endogonaceae. These fungi are obligate symbionts, and currently cannot be cultured i n the absence of a plant host (Powell and Bagyaraj 1984). . . -The mycorrhizal fungus, from a germinating spore or hypha of nearby roots, forms appressoria to penetrate the host epidermal c e l l s or root hairs. There may be multiple colonization points along the root. Once in s i d e the host root, the fungal hyphae spread throughout the c o r t i c a l c e l l s , but do not invade the endodermis, s t e l e or meristematic tissues of the host plant. Infection occurs most i n fin e , young roots. The two c h a r a c t e r i s t i c structures from which the name of t h i s mycorrhiza originates are formed by the fungus within the host root. MiEEiEiSS are ovoid or globose bodies 1 formed from swellings of hyphae i n older regions. They can be i n t e r c e l l u l a r or i n t r a c e l l u l a r , vary i n size, and may not be formed by a l l species of VA mycorrhizal fungi (Bonfante-Fasolo 1984). They contain large l i p i d droplets and are considered to be resting organs involved i n food storage. Arbuscules, resembling small bushes, are formed by the repeated dichotomous branching of the penetrating hyphae within the host c o r t i c a l c e l l s . They often nearly f i l l the host c e l l , providing an extremely large area of contact between the fungus and the cytoplasm of the host plant, and are considered to be the p r e f e r e n t i a l s i t e s for fungus/plant metabolite exchange (Scannerini and Bonfante-Fasolo 1983, Cox et a l . 1975). They are formed by a l l species of VA mycorrhizal fungi. The l i f e s p a n of an arbuscule i s from four to f i f t e e n days (Harley and Smith 1983), afte r which the branches deteriorate and collapse. VA mycorrhizae also have an associated extramatrical phase of thick- or thin-walled hyphae, the extent of which varies with the s o i l type, host plant and fungal symbiont <Bonfante-Fasolo 1984). Spores are usually associated with the external mycelium, but they may also be within the root. They may occur singly or within sporocarps. These spores are used to i d e n t i f y the fungal species involved i n VA mycorrhizae. Anything which can influence either the host plant or the fungal symbiont w i l l a f f e c t the formation of mycorrhizae. A major regulator of colonization of the host by the fungus i s s o i l phosphorus l e v e l , with high l e v e l s i n h i b i t i n g the formation of mycorrhizae (Cooper 1984). Other regulating factors, and examples of papers showing t h e i r e f f e c t s , include: s o i l moisture (Read and Boyd 1986), s o i l aeration (Saif 1981), pH (Wang et a l . 1985), temperature (Schenck and Smith 1982), heavy metals (Gildon and Tinker 1983), and fungicides (Nemec 1980). VA mycorrhizal fungi have been shown to promote the growth of t h e i r host plant, and t h e i r main influence i s thought to be on nutrient uptake. Although unable to u t i l i z e forms of nutrients d i f f e r e n t from nonmycorrhizal plants, mycorrhizal plants seem to have an altered a b i l i t y to absorb and translocate various elements (Tinker and Gildon . 1983). The extramatrical hyphae are believed to improve the exploratory geometry of the plant, extending beyond the rhizosphere to absorb nutrients which would otherwise be unavailable to the plant (Clarkson 1985). They are most important i n the uptake of nutrients such as phosphorus which move to the plant by d i f f u s i o n . Improved phorphorus uptake by VA mycorrhizae has been c l e a r l y shown by many examples of mycorrhizal plants growing well i n phosphate-deficient s o i l s i n which the growth of nonmycorrhizal plants was poor (Abbott and Robson 1984). Zinc and copper uptake, which i s also d i f f u s i o n - l i m i t e d , i s thought to be affected by VA mycorrhizae as well. However, i t i s d i f f i c u l t to ascertain whether mycorrhizae d i r e c t l y influence the uptake of these micronutrients or whether t h e i r increased uptake i s due to improved plant vigour from the increased phosphate supply to plants from the mycorrhizae (Abbott and Robson 1984). It has also been suggested that VA mycorrhizal fungi improve water uptake (Cooper 1984) and disease resistance (Bagyaraj 1984) i n t h e i r host plants. These are f e l t to be secondary e f f e c t s due to improved phosphorus n u t r i t i o n . Whether due to d i r e c t or secondary e f f e c t s , the benefits to be derived from VA mycorrhizae are of i n t e r e s t i n agriculture, e s p e c i a l l y to increase plant growth on i n f e r t i l e s o i l s (Abbott and Robson 1984). However, most investigations of mycorrhizae have been conducted i n pots i n greenhouses, and as t h e i r r e s u l t s are not always applicable to what happens i n f i e l d s , i t i s f e l t that more f i e l d research needs to be done ( F i t t e r 1985). Generally, the work described i n t h i s t h e s i s investigated VA mycorrhizae i n winter wheat under t y p i c a l f i e l d conditions i n South Coastal B r i t i s h Columbia, Canada. In the f i r s t part, the colonization patterns throughout the growing season at four d i f f e r e n t s i t e s were charted and were rela t e d to various f i e l d parameters. In the second part, the e f f e c t s of the systemic fungicides Bayleton and T i l t on colonization were investigated. 4 CHAPTER TWO COLONIZATION PATTERNS OF V.A. MYCORRHIZAL FUNGI ON WINTER WHEAT. In the past decade, winter wheat (Triticum aestivum L. ) has become a commonly grown crop i n temperate regions. It was introduced to South Coastal B r i t i s h Columbia to aid i n s o i l conservation by reducing s o i l degradation processes, and to provide a fall-seeded "cash" cover crop to farmers (Temple and Bomke 1989). As with any cash crop, i t i s desirable to maximize the y i e l d while minimizing the costs to obtain the greatest p r o f i t . Because f e r t i l i z e r c onstitutes a major portion of the cost of a crop to a farmer-, i t i s necessary to f i n d ways to decrease f e r t i l i z e r use, and to use more e f f i c i e n t l y that which i s applied. S o i l organisms, p a r t i c u l a r l y VA mycorrhizal fungi, are now being considered as " b i o t i c f e r t i l i z e r s " (Menge 1983), and studies are being conducted to e s t a b l i s h t h e i r a v a i l a b i l t i y and usefulness to various crops i n d i f f e r e n t regions. Although wheat has been shown to not be highly dependent on mycorrhizae (Young et a l . 1985), i n t e r e s t i n the VA mycorrhizae of winter wheat has been generated because these associations have been shown to improve the n u t r i t i o n and drought tolerance of some native grasses (Allen and Boosalis 1983) and spring wheat (Khan 1975, Hetrick and Bloom 1983, Jakobsen and Nielsen 1983) under low nutrient conditions i n areas where winter wheat i s also 5 grown. Of p a r t i c u l a r i n t e r e s t i s the length of time aft e r seeding required for plants to become colonized. Spring wheat becomes colonized very soon aft e r sowing and continues to be colonized throughout the growing season (Jakobsen and Nielsen 1983). It i s believed that seedlings are more dependant on colonization than are mature plants (St. John and Coleman 1983) and that VA mycorrhizal fungi may only s i g n i f i c a n t l y influence phosphorus uptake i n annual crops i f colonization i s established shortly a f t e r seedling emergence (Jakobsen and Nielsen 1983). An additional f a c t o r i n northern temperate regions i s s o i l temperature through the winter: i f colonization i s not established early on, conditions may not again be conducive to establishment u n t i l l a t e i n the spring, well past the c r i t i c a l seedling stage and too l a t e to provide much benefit to the wheat crop. Various researchers have examined VA mycorrhizae i n f i e l d crops of winter wheat i n temperate regions, and have produced c o n f l i c t i n g r e s u l t s . In Denmark (Jakobsen and Nielsen 1983) and i n Kansas, USA (Hetrick and Bloom 1983, Hetrick et a l . 1984) colonization was not found i n winter wheat u n t i l spring, and i t was concluded that VA mycorrhizae were not important to t h i s cereal crop. In contrast, researchers i n Nebraska, USA (Yocum et a l . 1985), Hertfordshire, England < Buwalda et a l . 1985b) and Kent, England (Dodd and J e f f r i e s 1986) observed l e v e l s of colonization ranging from 1'/. to 40% of the roots within two months of seeding. Others did not sample i n the f a l l , but report colonization i n the summer (Hayman 1970, Young et a l . 1985, Allen and Booealie 1983). Various theories have been put forward to explain the differences i n these re s u l t s , u t i l i z i n g what are believed to be the main influences on the general establishment of VA mycorrhizae i n winter wheat. These fact o r s may a f f e c t the mycorrhizal fungus, the host plant, or the two together i n the mycorrhiza. The most popular theory suggests that temperature i s the l i m i t i n g factor. It i s thought to be too low i n the f a l l to allow adequate growth of the fungus to the host, or of the host roots to the fungal inoculum, thus preventing the formation of the mycorrhiza (Jakobsen and Nielsen 1983, Hetrick and Bloom 1984, Young et a l . 1985, Buwalda et a l . 1985b). Inoculum density may also be a factor, acting analogously to temperature i n that the inoculum and roots may be too f a r apart f o r colonization to occur before winter (Jakobsen and Nielsen 1983, Hetrick and Bloom 1983). A t h i r d hypothesis i s that the chemical and physical properties of the s o i l i n h i b i t mycorrhiza formation (Salf and Khan 1975, Daniels and Trappe 1980, Hayman 1982, Jakobsen and Nielsen 1983, Young et a l . 1985, Buwalda et a l . 1985a, 1985b, Koske 1987). These properties include pH, moisture content, and nutrient content, a l l of which are considered to a f f e c t spore germination. As well, high s o i l phosphorus has been shown to decrease l e v e l s of root exudation, either reducing colonization or preventing i t e n t i r e l y (Graham et a l . 1981, Ocampo and Azcon 1985). Related to a l l of the previous theories i s the fourth, which proposes that inoculum s t r a i n and/or host c u l t i v a r decides the formation of mycorrhizae (Azcon and Ocampo 1981, Young et a l . 1985, Dodd and J e f f r i e s 1986). The experiments conducted to date on VA mycorrhizae i n winter wheat have a l l used d i f f e r e n t wheat c u l t i v a r s , and so differences i n t h e i r r e s u l t s could be due to genetic differences i n the plants, which cause them to react to environmental fac t o r s i n d i f f e r e n t ways. This i s also true f o r inoculum s t r a i n s . F i n a l l y , management practices and methods of t i l l a g e used by farmers i n the various regions studied could account for the v a r i a b i l i t y i n r e s u l t s (Jakobsen and Nielsen 1983, Yocum et a l . 1985, Young et a l . 1985). In l i g h t of the r e s u l t s and theories from previous research, the purpose of t h i s study was to investigate seasonal colonization patterns of VA mycorrhizae i n winter wheat at four s i t e s i n South Coastal B r i t i s h Columbia. To allow the application of some of the previous hypotheses on colonization to the r e s u l t s of t h i s study, many of the environmental fac t o r s believed to be influences were also monitored. 8 and Methods F i e l d S i t e s This study was conducted at four s i t e s i n South Coastal B r i t i s h Columbia. The s i t e s , named i n t h i s study for the nearest v i l l a g e or town to each, are: Oyster River at the University of B r i t i s h Columbia Oyster River Research Farm (49°55'lat., 125°10'long.); Delta at Reynelda Farms on Westham Island (49°10', 123°10')j Chilliwack on the Frank's farm (49°10', 121°50'); and Agassiz at the Agriculture Canada Agassiz Research Station <49°15', 121°45') (west to east, Fig. 1). These s i t e s were established as part of a project i n v e s t i g a t i n g intensive cereal management i n the region (Temple and Bomke 1989). A general description of each s i t e can be found i n Table I. Oyster River, with a very well drained loamy sand s o i l , was the d r i e s t of the four s i t e s and was the least prone to disease. The poorly drained s i l t loam s o i l at Chilliwack made i t the wettest s i t e . Agassiz had the most severe incidence of disease. The crops planted on these s i t e s p r i o r to t h i s study d i f f e r e d ; Oyster River was the only s i t e which was previously i n winter wheat. The general layouts of the areas sampled are shown i n the photographs of the four s i t e s i n May 1988 i n Fig. 2. The four s i t e s were a l l subject to the same management practices (Table I I ) . Each s i t e was seeded with winter wheat (Triticum aestivum L. cv Monopol) at a rate of 150-200 kg/ha, using a 9 cm row spacing. The R seeds were treated with Vitavax (carboxin, UniRoyal Chemical Ltd.) according to the manufacturer's 9 Vancouver AGASSIZ, CHILLIWACJ INDEX MAP 1 N i U . 9 o 0 0 . . . . N 1 2 3 ^ 0 0 ' Canada U.S.A. Ei9HE§- i : A m a P o f the f i e l d s i t e s used i n t h i s study. indicates a study s i t e , while -^Jf indicates a town or c i t y , depending on si z e . I l b l e I: Descriptions of the f i e l d s i t e s . I General Descrigtion S i t e Oyster River Agassiz Chilliwack Delta loamy sand; very well drained; low.disease incidence; subject to drought; previous crop: winter wheat. s i l t loam; moderately well drained; very severe disease incidence; moderate water holding capacity; previous crop: clover. s i l t loam; poorly drained; severe disease incidence; moderate water holding capacity; previous crop: bush beans. s i l t y clay loam; under drained; low-moderate disease incidence; good water holding capacity; previous crop: potatoes. 10 s p e c i f i c a t i o n s . The dates of planting can be found i n Table III. The growth stages to which Tables II and III re f e r are i n the Zadok or decimal code (Tottman and Makepeace 1979). The Oyster River s i t e did not receive the second fungicide R treatment of T i l t (Ciba-Geigy Ltd.) at growth stage 34-37. Tramlines were established at each s i t e , and the treatments of Table II were applied using farm machinery (Temple and Bomke 1989). In addition, herbicides were applied at three of the s i t e s at approximately growth stage 30. These were: R Blagal (cyanazine and MCPAK, Ciba-Geigy Ltd. ) at R Chilliwack; Mecaprop (May and Baker Canada Ltd.) at Oyster R River, and Glean (chlorsulfuron, Dupont Canada Inc.) at Agassiz. These were a l l applied according to the manufacturers' s p e c i f i c a t i o n s . The Delta s i t e did not receive an herbicide treatment. Samgling Methods Table III shows the dates on which the f i v e harvests were taken at each s i t e . The f i r s t two dates were selected because they occurred before and aft e r the coldest of the winter temperatures had been experienced at these s i t e s . Harvest A (known as the December harvest from here on) at Oyster River was l a t e r than the f i r s t harvests at the other s i t e s because planting there was delayed by very dry conditions. The remaining three harvest dates were selected based on the growth stage o f r t h e plants: Harvest C (May) corresponded to stem elongation, Harvest D (June) to anthesis and Harvest E (August) to the f i n a l crop harvest 12 Table I I : Crop treatments. Treatment Growth Stage F e r t i l i z e r as Urea (kg/ha N) 50 22 125 31 50 37 Plant Growth Regulator "Cycocel" 3 L/ha 31 Fungicide "Bayleton" 250 g/ha 32 " T i l t " 0.5 L/ha 34-37 " T i l t " 0.5 L/ha 55 I l ^ i e Dates of planting and harvesting at each s i t e , and the Zadok growth stage (GS) at each harvest. Planted Harvest A GS Harvest B GS Harvest C GS Harvest D GS Harvest E GS Oyster River 16-10-87 10-12-87 11-19 26-02-88 19-21 18-05-88 35 18-06-88 62-66 04-08-88 80-92 SITE Agazzis 30-09-87 17- 11-87 14-21 18- 02-88 16-33 05-05-88 33 12-06-88 59-65 01-08-88 85-92 Chilliwack 29-09-87 17- 11-87 15-22 18- 02-88 18-24 05-05-88 34 12-06-88 62-66 01-08-88 90-92 Delta 01-10-87 24-11-87 16-24 29-02-88 N/A 11-05-88 31-32 24-06-88 65-68 17-08-88 80-92 13 when the moisture content of the grain had reached 177. (Temple and Bomke 1989). In Figure 3 there are photographs from Oyster River which are representative of the plants at the f i v e harvests at a l l s i t e s but the February harvest at Delta. Unfortunately, i n l a t e December the s i t e was grazed by wigeons (Anas penelope L. and Anas amerlcana L. ), which clipped a l l of the leaves from the young plants. This prevented the c o l l e c t i o n of f o l i a r samples i n February from that s i t e (Fig. 4). The root systems remained intact, however, and the plants were f u l l y recovered by the May harvest (Fig. 2). For the f i r s t three harvests, the en t i r e plant was removed. The f o l i a g e was clipped and then the roots were dug up with some of the surrounding s o i l to a depth of 20 cm (Fig. Sa). The number of plants sampled was generally determined by the amount of f o l i a g e required for analysis; i n i t i a l l y about 40 plants were taken, but as the plants grew, fewer were needed. Four r e p l i c a t e samples were taken each time from each s i t e , and approximately the same areas of the s i t e s were sampled at each harvest. As the root systems of the plants became more extensive, the sampling methods were changed, as was reported by Buwalda et a l . (1985b). For the l a s t two harvests, the shoots of ten plants per r e p l i c a t e were clipped, and two cores were taken at the base of each of the clipped plants to a 20 cm depth (Fig. 5b, 5c). After a l l harvests, the samples were kept at about 5°G for no more than 24 hours, u n t i l they could be preserved. Ei9y£§? 3 : Representative photographs of the plants at the f i v e sampling dates used i n t h i s study. (Pictures taken at the Oyster River s i t e . ) C(a)December <b)February (c)May (d)June (e)August] 15 16 Figure 4: A photograph of the Delta s i t e at the February harvest. Sample Preservation In the laboratory, the roots were separated from the s o i l by sieving, and non-wheat roots were discarded. The roots were washed and fixed i n a 90:5:5 formaldehyde:acetic acid-.ethanol (FAA) solution (modified from P h i l l i p s and 17 Ei9y.££ 5: Photographs of the sampling methods for the f i r s t three harvests (a) and for the l a s t two harvests (b and c >. 18 Hayman (1970)). The s o i l was a i r dried, sieved to < 2mm, and stored i n p l a s t i c bags. The f o l i a r samples were checked for cleanliness, and d i r t y leaves were discarded. The f o l i a g e was then ovendried at 60 ° C for 12-36 hours, depending on the sample size, ground with a Wiley m i l l to < 2mm, and stored i n p l a s t i c v i a l s . L§99r.lfQ**Y. Analysis The root samples were cleared and stained with a modified version of the P h i l l i p s and Hayman (1970) method. After c l e a r i n g f o r one hour at 90°C i n 10% KOH, they were cooled, rinsed, a c i d i f i e d with l a c t i c acid, and stained i n 0.1% (W/V) Trypan blue s t a i n i n a 1:1:1 solution of g l y c e r o l : l a c f i c a c i d : d i s t i l l e d water at room temperature for 24 hours. The excess s t a i n was then drained, and the samples were l e f t i n a destaining s o l u t i o n of 1:1:1 g l y c e r o l : l a c t i c a c i d : d i s t i l l e d water at room temperature. The samples were l a t e r stored i n destaining s o l u t i o n i n a i r t i g h t p l a s t i c v i a l s . P r i o r to counting, the roots were cut into 1 to 2 cm pieces. Subsamples of about 10 ml of roots i n destaining solution were spread evenly i n more destaining s o l u t i o n over the bottom of a P e t r i plate, and were counted as per Giovanetti and Mosse (1980), using a 1.25 cm by 1.25 cm g r i d and 25x magnification (Fig. 6). One hundred root pieces were counted per subsample, and six subsamples were taken for each of the duplicate v i a l s of the four r e p l i c a t e samples per harvest per s i t e ( r e s u l t i n g i n 24 counts/site/date). 19 Ei9y.?C§i § J Methods by which counts of percent colonization were done. Spores were extracted from the December and August s o i l samples using sucrose centrifugation (Smith and Skipper 1979). These dates were chosen to represent the s t a r t and end of the growing season. I d e n t i f i c a t i o n s were made by Dr. S. M. Berch, University of B r i t i s h Columbia. S o i l pH was determined using a 1:1 soil:water mixture (McLean 1982) with an analog pH meter (Orion Research). The Mehlich 3 method (Mehlich 1984) was used to determine s o i l phosphorus, copper and zinc. Samples were read using an Inductively Coupled Plasma (ICP) instrument (Fisher S c i e n t i f i c Co.). A second s o i l phosphorus analysis for comparative purposes was made using the modified Bray PI technique (Bray and Kurtz 1945, Watanabe and Olsen 1965) and a Spectronic 20 spectrophotometer (Bausch and Lomb). F o l i a r 20 phosphorus, copper and zinc were extracted with Parkinson and Allen digests (Parkinson and Allen 1975) and were read on the ICP instrument. Climate Information Ai r temperature and p r e c i p i t a t i o n information for the 1987-1988 growing season was obtained from the Vancouver o f f i c e s of Environment Canada. S o i l temperatures were not measured at the f i e l d s i t e s used during t h i s study, but "normal" s o i l temperatures (Ouellet et a l . 1975) were used instead. These have been determined using models and c l i m a t i c data c o l l e c t e d f o r between 15 and 3(3 years. Temperatures for Comox and Steveston were used f o r the Oyster River and Delta s i t e s , respectively, as they were the nearest locations to these s i t e s f o r which normal temperatures were available. These temperatures are not intended to give an exact representation f o r the s o i l temperatures of the four f i e l d s i t e s during the 1987-1988 growing season, but instead are included f o r comparison to temperatures reported by researchers i n other countries. Thermal time f o r each s i t e was determined by. subtracting the mean d a i l y a i r temperature from a base temperature (5° C), and then adding a l l of the p o s i t i v e r e s u l t s , as per Buwalda et a l . (1985b). S t a t i s t i c a l Analysis S t a t i s t i c a l analyses were done, using the Genlin programme (The University of B r i t i s h Columbia) to perform 21 analysis of variance and Tukey's (HSD) Tests. Prio r to analysis, the r e s u l t s from the colonization studies were transformed using log <Y.I * 1), where V.1 i s percent colonization <St. John and Hunt 1983, Buwalda et a l . 1985b). Log transformations were done to the f o l i a r zinc data, while log <x • 1) transformations were done to the r e s u l t s from the s o i l copper and. f o l i a r phosphorus r e s u l t s (where x i s the r e s u l t obtained) (Devore 1982). Pearson pairwise c o r r e l a t i o n s were done with the Mystat software package on an AST personal computer. 22 f Results Climate Information Table IV shows the mean monthly a i r temperatures and p r e c i p i t a t i o n at the four f i e l d s i t e s during the study. In the f a l l . Oyster River was the coolest of the s i t e s (14.2 -5.a°C), while Chilliwack was the warmest (17.0 - 7.5°C>. During December and January, the Agassiz and Chilliwack s i t e s experienced the coldest temperatures (down to 1.9 and 0.9°C re s p e c t i v e l y ) , but for the remainder of the growing season warmed up quickly and, with Delta, became the warmest s i t e s , with high temperatures of about 18°C). The a i r temperatures at Oyster River were the coolest through the spring and summer. Generally, Chilliwack received the most r a i n f a l l , e s p e c i a l l y from December through May, with t o t a l p r e c i p i t a t i o n of 1479 mm for those s i x months. Delta received the lea s t p r e c i p i t a t i o n . The s o i l temperature trends (Table V) are s i m i l a r to those f o r a i r temperature, but differences among the s i t e s are les s pronounced, and the winter temperatures are not as cold, with a low of only 2.9°C. Colonization Counts and Spore I d e n t i f i c a t i o n s The colonization patterns obtained i n t h i s study, are displayed i n Figure 7. There was about 1'/. colonization at the Oyster River and Agassiz s i t e s at the December harvest (Table VIb). These low but appreciable l e v e l s of colonization persisted through February, and then increased 23 Table IV: Mean monthly a i r temperatures (C) and p r e c i p i t a t i o n (mm), Sept.1987 to Aug. 1988. SITE Qzster RiVi. Agassiz Chilliwack Delta Month TernBi. Temfii. ESBi. Temg._ Pcg._ Temgj_ Pep. Sept. 14. 2 25. 4 16. 5 44. 6 17. 0 66. 2 15. 2 18. 0 Oct. 8. 8 31. 0 12. 1 27. 0 12. 5 35. 2 9. a 26. 4 Nov. 5. a 204. a 8. 1 147. 7 7. 5 181. 9 8. 1 117. 4 Dec. 2. a 216. 4 2. 3 212. 2 1.6 292. 0 3. 4 145. 1 Jan. 2. 9 187. 7 1. 9 142. 7 0. 9 221. 0 3. 2 80. 2 Feb. 4. 2 76. 7 5. 0 148. 6 4. 8 194. a 5. 3 60. 2 Mar. 5. 6 140. 2 7. 1 241. 4 6. 7 325. 6 6. 9 121. 2 Apr. 8. 4 79. 2 10. 2 193. 6 10. 0 255. a 10. 0 94. 3 May 10. 9 63. 1 13. 2 169. 1 13. 4 192. 6 12. 7 127. 1 June 13. 9 40. a 15. 2 61. 7 15. 6 66. 2 15. 3 36. 0 July 16. 9 25. 6 18. 0 79. a 18. 5 119. 4 18. 0 31. 0 Aug. 16. 9 21. 4 18. 0 37. 6 18. 5 64. a 17. 6 27. 2 I§.hle V: Normal s o i l temperatures <C), September to August, at a depth of 10 cm. SITE Oyster Riv._ Agassiz Chilliwack Delta Month Tempi. Temp.. Temp.. Temg._ Sept. 15. 5 16. 5 16. a 15. 5 Oct. 11. 0 13. 0 13. 0 11. 2 Nov. 7. 0 8. 0 8. 0 7. 0 Dec. 4. 3 4. 9 4. 7 4. 4 Jan. 3. 2 3. 2 2. 9 3. 5 Feb. 4. 0 4. 4 4. 4 4. 4 Mar. 5. 6 6. 2 5. 9 6. 4 Apr. 9. 5 11. 9 11. 5 11.0 May 14. 3 15. a 15. a 15. 0 June 17. 6 18. 6 18. a 17. 3 July 19. 7 20. 6 i a . 8 17. a Aug. 17. 9 18. 9 19. 1 17. 6 24 c o cC N o o u rt o <D PH December February May June August H a r v e s t D a t e ( D e c e m b e r 1987 - A u g u s t 1 988 ) Ei9iJEe 7: Colonization patterns f o r each s i t e through time. (I i s the standard error of the means). Table Via: Analysis of variance table for colonization. Sum of Mean Source Sguares BE F-ratig Probability Site 29. 14 3 9. 71 436.46 0. 000 Date 70. 89 4 17. 72 796.34 0. 000 Site»Date 28. 98 12 2. 42 108.S3 0. 000 Error 10. 24 460 0. 02 Total 139.25 479 TahiS VIb: Mean colonization at each s i t e through the growing season (percent). SITE Date Qx<§ter River Agassiz Chilliwack Delta Dec. 1 c» 1. c 0 a 0 a Feb. 2 c l b c 0 a 0 a Hay 21 g 13 f 0 a 0 a June 32 h 22 gh 4 d 17 fg Aug. I c 4 d 0 ab 8 e » Those followed by different letters d i f f e r significantly using Tukey's <HSD) Test (p < 0.05). 25 by May to 21'/. for Oyeter River and 137. for Agassiz. The Chilliwack plants were not colonized at a l l during those three harvests, and colonization was only very s l i g h t by May at Delta (Table VIb). The June harvest showed peak l e v e l s of between 47. and 32% f o r a l l four s i t e s , which dropped for the f i n a l harvest i n August. The colonization l e v e l s were highest at Oyster River for every harvest but the l a s t , and were consistently lower at Chilliwack. S t a t i s t i c a l l y , s i t e , date and site*date were s i g n i f i c a n t at p < 0.05 (Table Via), as were most of the differences among the s i t e s (Table VIb). Figure 8 shows the same colonization r e s u l t s as Figure 7, but these are plotted against thermal time rather than date. Colonization at Oyster River was established before 500°C days, peaked at 1000°C days, and had declined by 1500° C days. These are much lower temperatures than those f o r the other three s i t e s , which peaked at about 1500°C days and declined between 1500 and 2000°C days. The thermal times fo r i n i t i a l c olonization at Oyster River and Agassiz were similar, but were much cooler than the thermal times at which colonization was f i r s t observed at Delta and Chilliwack. In Figures 9 and 10 are pictures of colonized roots. These show colonization patterns which are representative, of those seen at Oyster River and Agassiz through the growing season, and Delta i n the l a t e r stages. Colonization was only seen i n Chilliwack roots i n June, and resembled early stages at the other s i t e s . Early colonization (Fig. 9a) was sparse i n the roots. Young arbuscules are v i s i b l e , and 26 50 PI o • ^ -t-> «s N Pi o o a +j PI CO o 0) CL, 40 o - Oyster R iver O — Agass iz o - C h i l l i w a c k a - De l ta 500 1000 1500 Thermal Time (° C) 2000 Figure 8: Colonization patterns i n thermal time (degree days over 5 C). appressoria can be seen, although they are not shown i n t h i s picture. There was no change i n the appearance of the mycorrhizas from December to February. In the spring, the roots became more densely colonized (Fig 9b). With time, the arbuscules decreased, and vesicules were formed (Fig 9c). Eventually, as the roots began to die, the mycorrhiza became mainly vesicular (Fig. 9d). Both coarse and f i n e endophyte were present at the s i t e s , although Agassiz was dominated by fin e , and Oyster River by coarse (Fig. 10). 27 Ensure 9 : Photographs of colonized roots, displaying the general colonization patterns observed over time. [ ( a ) f a l l , 537.5x mag. (b)spring, 1850x mag. (c)midsummer,462.5x mag. (d)late summer, 462.5x mag.] 28 29 There were few s i m i l a r i t i e s among the s i t e s i n terms of th e i r fungal species (Table VII, Figure 11). S c l e r g c y s t i s rubiformis was found at Oyster River, Delta (August) and Chilliwack (August), but not at Agassiz, while Glomus balonatum was seen at both Delta and Agassiz, but not at the other s i t e s . At a l l s i t e s but Agassiz, the species found during December were, along with other species, also found i n August. The Agassiz s i t e i n December had the greatest number of species of any harvest of the s i t e s , but none of these species were present i n the August samples. S o i l Analyses The two methods of analysis for s o i l phosphorus produced f a i r l y s i m i l a r r e s u l t s (Figures 12 and 13). The Chilliwack and Delta s o i l s had very high l e v e l s of phosphorus (100-150 mg/kg), which were s i g n i f i c a n t l y d i f f e r e n t from those at Agassiz and Oyster River (30-90 mg/kg) (Tables VHIb, IXb). Both a n a l y t i c a l methods showed a pronounced decrease at Delta i n June. S t a t i s t i c a l l y , the Bray PI method produced s i g n i f i c a n t s i t e , date and site*date differences (p < 0.05) (Table V i l l a ) , while only the s i t e s were s i g n i f i c a n t l y d i f f e r e n t with the Mehlich 3 method (Table IXa). 30 Table VII: Fungal species I d e n t i f i e d from the study s i t e s . The numbers ref e r to voucher c o l l e c t i o n s held by Dr. S. M. Berch, as per Appendix II. S i t e Date Fungus Oyster Dec. Glomus mgngsQgrum Gerdemann & Trappe River S c l e r g c y s t i s rubifgrmis Gerdemann & Trappe Aug. Glomus sp. 73 Glomus mgngsporum Gerd. & Trappe S c l e r g c y s t i s rubifgrmis Gerd. & Trappe Agassiz Dec. Glomus sp. 87 Glomus sp. 91 Glomus halgnatum Rose & Trappe Scutellgsp.gra sp. 89 Aug. Acaulgsggra sp. 86 Chilliwack Dec. Glgmus SP* Q"7 Aug. Acaulgsggra sp. 79 Glomus sp. 87 Sc l e r g c y s t i s rubifgrmis Gerd. & Trappe Delta Dec. Glgmus halgnatum Rose & Trappe Aug. Glgmus halgnatum Rose & Trappe S c l e r g c y s t i s rubifgrmis Gerde & Trappe 31 (a) (b) Figure 11: Fungal spores from the s i t e s . C(a)Acaylosggra sp. 86, 2175x; < b> Agaulospora sp. 79, 2175x; <c)Sclerocystis rubifgrmis, 1362x; (d)Scutellospora sp. B9, 2175x; <e) Glgmus sp. 73; 1362x; (f)G._ halgnatum, 2175x; (g)G._ mgngeggr urn, 2175x; (h)Glgmus sp. 91, 1362x; (DDlgmuB sp. 87, 1362x. (c) (d) 32 200 M \ a, cn *h o Xi ft ai O On O w .—I Figure 12 150 100 -50 - • - Oyster R iver O - Agass iz o - C h i l l i w a c k A - De l ta December February May June August Harvest Date (December 1987 - August 1988) S o i l phosphorus <Bray PI) per s i t e through time. (I i s the standard error of the means. > Table V i l l a : . Analysis of variance table f o r Bray PI s o i l phosphorus. Sum of Mean Source Sguares DF Sguares F-ratio Prgbabili Site 58628.0 3 19543.0 50.043 0. 000 Date 7961..1 4 1990.3 5. 097 0. 001 Site »Date 13232.0 12 1102.7 2. 824 0. 004 Error 23431.0 60 390.52 Total 103250.0 79 Table VIIlb: Mean avail a b l e s o i l phosphorus (Bray PI) at each s i t e through the growing season (mg/kg). SITE Date Qyster River Agassiz Chilliwack Delta Dec. 78.0ab» 84.lab 156.3ef 102.Sabcd Feb. 70.4a 76.7ab 169.If 139.4cdef May 72.7a 101.9abcd 156.5ef 126.0bcdef June 86.3ab 79.9ab 148.2def 107.2abcde Aug. 71.1a 94.9abc 104.0abcd 78.0ab * Those followed by different letters d i f f e r significantly using Tukey's (HSD) Test (p < 0.05). 34 60 CO O ft CO o DH o CO CO o •iH CP 200 150 100 50 Figure 13: I • — O y s t e r R i v e r o — A g a s s i z o ~ C h i l l i w a c k A - D e l t a December F e b r u a r y May June August Harvest Date (December 1987 - August 1988) S o i l phosphorus <Mehlich 3) per s i t e through time. (I i s the standard error of the means.) Table IXa: Analysis of variance tables f o r Mehlich 3 s o i l phosphorus. Sum of Mean Source Sguares BE Sguares Ezratig Probability Site 1 7 9 6 8 0 . 0 3 5 9 8 9 3 . 0 9 2 . 4 1 3 0 . 0 0 0 Date 3 3 4 4 . 2 4 8 3 6 . 1 1 . 2 9 0 0 . 2 8 4 Site»Date 1 3 6 8 9 . 0 12 1 1 4 0 . a 1 . 7 6 0 0 . 0 7 6 Error 3 8 8 8 6 . 0 60 6 4 8 . 1 Total 2 3 5 6 0 0 . 0 79 Table IXb: Mean availa b l e s o i l phosphorus (Mehlich 3) at each s i t e through the growing season (mg/kg). SITE Date Oyster River Agassiz Chilliwack Delta Dec. 52.0ab» 40.7a 150.2c 124.2c Feb. 45.1a 31.8a 179.0c 155.8c May 55.3ab 46.3a 123.6c 162.3c June 53.7ab 29.4a 130.4c 129.3c Aug. 45.5a 55.4ab 128.8c 116.4bc » Those followed by different letters d i f f e r s i g n i f i c antly using Tukey's <HSD> Test (p < 0.05). 35 The Oyster River s o i l contained the highest l e v e l s of zinc (about 2 mg/kg) (Figure 14), while Agassiz contained the lowest with close to 1 mg/kg (Table Xb). The zinc l e v e l s at Oyster River were r e l a t i v e l y stable throughout the growing season, while those, at the other s i t e s generally increased. The sharp increase i n June i n the Chilliwack s o i l (from 1 to 2 mg/kg) may be due to contamination from using a brass sieve for those samples (Lavkulich 1982). Table Xa indicates that s i t e , date and site»date were s i g n i f i c a n t (p < 0.05). S o i l copper was highest at Delta (6-9 mg/kg) and lowest, at Agassiz (1-3 mg/kg) (Figure 15, Table XIa). A l l s i t e s had s i g n i f i c a n t increases i n June, which dropped again i n August. Again, s i t e , date and site»date were s i g n i f i c a n t . The s o i l s at a l l of the s i t e s used i n t h i s study had pH readings of le s s than pH 6, and showed a general decrease i n pH over time (Fig. 16). Site, date and site»date differences were s i g n i f i c a n t (p < 0.05) (Table X l l a ) . The Delta s o i l pH was s i g n i f i c a n t l y higher (Table Xllb) than those of the others i n December (pH 5.7), but at about pH 5.0 was not very d i f f e r e n t from Oyster River for. the remainder of the harvests. The pH at Chilliwack was the lowest at every harvest, and was always l e s s than pH 4. 8. 36 &0 W) o o el B 2 o CO I D — O y s t e r R i v e r O — A g a s s i z o - C h i l l i w a c k A - D e l t a 1 1 August December F e b r u a r y May June Harvest Date (December 1987 - August 1988) Figure 14: S o i l zinc per s i t e through time. (I i s the standard error of the means.) Table Xa: Analysis of variance table for s o i l zinc. Sum of Mean Source Sguares DF Sguares F-ratio Probability Site 0.642 3 0.214 37.393 0.000 Date 0.270 4 0.068 11.793 0.000 Site»Date 0.193 12 0.016 2.811 0.004 Error 0.343 60 0.006 Total 1.449 79 Table Xb: Mean availa b l e s o i l z inc at each s i t e through the growing season (mg/kg). SITE Date Oyster River Agassiz Chilliwack Delta Dec. 2.04cdef• 0.91ab 1.llabcd 0. 76a Feb. 2. 56f 0. 62a 0.91ab 1.42abcdef May 2. 55f 0. 80a 1.20abcde 1.91bcdef June 2. 63f 0.9Sabc 2. 47f 2.35def Aug. 2.36ef 1.42abcdef 1.88bcdef 2.08cdef » Those followed by different letters d i f f e r s i g n i f i c a n t ly using Tukey's (HSD) Test (p < 0.05). 37 10 \ P« o o CO 4 -2 -• — O y s t e r R i v e r O — Agass i z o — C h i l l i w a c k A - D e l t a December F e b r u a r y May June August Harvest Date (December 1987 - August 1988) Eisyr® 15' S o i l copper per s i t e through time. (I i s the standard error of the means.) Table XIa: Analysis of variance table f o r s o i l copper. Table Xlb: Sum of Mean Source Sguares BE Sguares Ezratlg Probability Site 359.61 3 119.87 156.82 0. 000 Date 48. 91 4 12. 23 16. 00 0. 000 Site»Date 24. 55 12 2. 05 2. 67 0. 006 Error 455.86 60 0. 77 Total 478.94 79 Mean s o i l copper at each s i t e through the growing season < mg/kg). SITE Date Oyster River Agassiz Chilliwack Delta Dec. 2.52ab» 2,34ab 3.07ab 7.88cd Feb. 3.Blab 1. 01a 2.60ab 6.78cd May 2.63ab 1. 32a 2.44ab 6.73cd June 3.18ab 3.01a 6. 66c 8. 95d Aug. 2.95ab 2.09ab 4. 17b 7.24cd • Those followed by different letters d i f f e r significantly using Tukey's (HSD) Test (p < 0.05). 38 6.4 -6.0 5.6 -5.2 O IT! 4.8 4.4 4.0 3.6 • - O y s t e r R i v e r o — A g a s s i z o - C h i l l i w a c k A - D e l t a December F e b r u a r y May June August Harvest Date (December 1987 - August 1988) Eiayre 16: S o i l pH per s i t e through time. (I i s the standard error of the means.) Table X l l a : Analysis of variance table for s o i l pH. Sum of Mean Source Sguares DF Sguares F-ratig Probability Site 4.880 3 1.627 46.366 0.000 Date 3.447 4 0.862 24.561 0.000 Site»Date 1.276 12 0.106 3.032 0.002 Error 2.105 60 0.035 Total 11.708 79 Table X l l b : S o i l pH at each s i t e through the growing season (means). SUE Date Qyster River Agassiz Chilliwack Delta Dec. 5.08ef» 5.15ef 4.75bcde 5. 70g Feb. 5.15ef 4.73bcde 4.55abc 5.28fg May 5.05def 4.73bcde 4.43ab 4.85bcdef June 4.78bcde 4.73bcde 4.38ab 4.70bcde Aug. 4.75bcde 4.58abcd 4.10a 4.98cdef » Those followed by different letters d i f f e r significantly using Tukey's (HSD) Test (p < 0.05). 39 F o l i a r Analysis F o l i a r phosphorus decreases with time, from 2-3 g/kg when the plants were young and green to 1 or l e s s as the plants became straw and grain (Fig. 17). Table XUIa shows that, s t a t i s t i c a l l y , s i t e , date and slte*date were a l l s i g n i f i c a n t (p < 0.05). In December, the Oyster River f o l i a r phosphorus l e v e l s were s i g n i f i c a n t l y higher than the others at 2.9 g/kg (Table XHIb) but were the lowest by August (0.4 g/kg). There were no s i g n i f i c a n t differences i n the phosphorus l e v e l s among the s i t e s i n February, May or June. As Figure 18 c l e a r l y shows, the f o l i a r zinc r e s u l t s ' were quite variable, but were s t a t i s t i c a l l y s i g n i f i c a n t (Table XlVa). The Oyster River l e v e l s were highest i n December (19.0 mg/kg) and lowest i n August (5.7 mg/kg) (Table XlVb), but Agassiz had the highest f o l i a r zinc readings of the February harvests. At a l l s i t e s , z i n c l e v e l s decreased i n May and June, but those from Chilliwack and Delta rose again i n August from 5.9 and 7.9 mg/kg to 14.0 and 13.0 mg/kg respectively. The f o l i a r copper l e v e l s i n plants from Agassiz and Chilliwack increased from December to February (1.9 and 2.8 mg/kg to 3.1 and 3.6 mg/kg), while those at Oyster River decreased s l i g h t l y from 4.1 to 3.8 mg/kg (Figure 19). Levels at a l l s i t e decreased from February to plateaux from May to August. However, these plateaux are due to the i n a b i l i t y of the ICP machine to di s t i n g u i s h l e v e l s below 1 mg/kg, and the f o l i a r copper l e v e l s i n r e a l i t y most l i k e l y 40 4 . 0 \ bo cn P o ft cn O 4 3 PH SH CO 'o 3 . 0 -2 . 0 1.0 0 . 0 • - O y s t e r R i v e r O - A g a s s i z o - C h i l l i w a c k A - D e l t a December February May June August H a r v e s t D a t e ( D e c e m b e r 1 9 8 7 - A u g u s t 1 9 8 8 ) EiayEg! i Z : F o l i a r p h o s p h o r u s p e r s i t e t h r o u g h t i m e . ( T h e F e b r u a r y h a r v e s t a t D e l t a w a s u n a v a i l a b l e . ) ( I i s t h e s t a n d a r d e r r o r o f t h e m e a n s . ) Table X J I J a : A n a l y s i s o f v a r i a n c e t a b l e f o r f o l i a r p h o s p h o r u s . T a b l e X H I b : Source Site Date Site»Date Error Total Sum of Sguares 0. 022 0. 984 0. 097 0. 064 1. 159 BE 3 4 11 57 75 Mean Sguares 0. 008 0. 246 0. 009 0. 001 F-ratio Probability 6. 84 220.78 7. 89 0. 001 0. 000 0. 000 M e a n f o l i a r p h o s p h o r u s a t e a c h s i t e t h r o u g h t h e g r o w i n g s e a s o n ( g / k g ) . Date Dec. Feb. May June A u g . SITE Oyster River Agassiz Chilliwack 2.93e» 2.44ghi 1.52de 1. 28d 0. 39a 2. 24fgh 2.52ghi 1.66def 1.54de 1.19cd 2.67hi 2.50ghi 1.57de 1. 42d 0.64ab Delta 2.45ghi n/a 1.99efg 1.53de 0.85bc » Those followed by different l e t t e r s d i f f e r s i g n i f i c a n t l y using Tukey's (HSD) Test (p < 0.05). n/a: The Feb. harvest at Delta was unavailable. 41 O C •rH tsa In Cfl •rH i— I o F i g u r e 1 8 24 20 16 12 8 -4 -• - Oyster River O - Agassiz o - Ch i l l iwack A - Delta December February May June August Harvest Date (December 1987 - August 1988) F o l i a r z i n c p e r s i t e t h r o u g h t i m e . ( T h e F e b r u a r y h a r v e s t a t D e l t a i s u n a v a i l a b l e . ( I i s t h e s t a n d a r d e r r o r o f t h e m e a n s . > T a b l e X I V a : A n a l y s i s o f v a r i a n c e t a b l e f o r f o l i a r z i n c . Sum of Source Sguares BE Site 0. 046 3 Date 1. 127 4 Site*Date 0. 664 11 Error 0. 195 57 Total 1. 994 75 an uaree EzEStio Probability 0.115 4.50 0.007 0.282 82.47 0.000 0.060 17.67 0.000 0. 034 a b l e X I V b : M e a n f o l i a r z i n c a t e a c h s i t e t h r o u g h t h e g r o w i n g s e a s o n ( m g / k g ) . SITE Date Oygter River Agassiz Chilliwack Delta Dec. 19.02h* 10.36def 11.10ef 11.00ef Feb. 13.36fg 18.35gh 12.37f n/a May 7.37abcd 6.92abc 7.18abc 8.19bcde June 6.40abc 8.04bcde 5.88ab 7.87abcde Aug. 5.70a 8.37cde 13.97fgh 12.99fg » Those followed by different letters d i f f e r s i g n i f i c a n t l y using Tukey's (HSD) Test (p < 0.05). n/as The Feb. harvest at Delta was unavailable. 4 2 6 \ W) <D ft OH O O - H <8 •i-H r—t o P*H 3 -• - Oyster R iver O - Agass iz o - C h i l l i w a c k - De l ta December February May June August Harvest Date (December 1987 - August 1988) Eisy-Ci! i ? J F o l i a r copper per s i t e through time. (The February harvest at Delta was unavailable (I Is the standard error of the means.) iMhle? XVa: Analysis of variance table f o r f o l i a r popper. Sum of Mean Source Sguares DF Sguares F-ratio Probability Site 5.01 3 1.67 12.33 0.000 Date 72.30 4 18.07 133.49 0.000 Site»Date 10.34 11 0.94 6.94 0.000 Error 7.72 57 0.14 Total 92.97 75 Table XVb: Mean f o l i a r copper at each s i t e through the growing season ,(mg/kg). SITE BSte Qy.ster River Agassiz Chilliwack Delta Dec. 4.07e» 1.87abc 2.80cd 3.32de Feb. 3.75de 3.08d 3.56de n/a May 1.38ab 1.21ab 1.08ab 1.98bc June 1.00a 1.61ab 1.00a 1.86abc Aug. 1.00a 1.00a 1.00a 1.00a » Those followed by different letters d i f f e r s i g n i f i c a n t l y using Tukey's (HSD) Test (p < 0.05). n/a: the Feb. harvest for Delta was unavailable. 43 dropped i n a l i n e a r fashion as occurred with f o l i a r phosphorus (Fig. 17). The r e s u l t s of t h i s analysis were s t a t i s t i c a l l y s i g n i f i c a n t (p< 0.05) (Table XVa), as were the more obvious differences among the s i t e s (Table XVb). Correlations The f o l i a r elements are a l l p o s i t i v e l y correlated to one another, and none correlates with Mehlich 3 s o i l phosphorus (Table XVI). F o l i a r phosphorus i s correlated p o s i t i v e l y to Bray PI s o i l phosphorus and s o i l pH, and negatively to s o i l zinc. F o l i a r copper i s p o s i t i v e l y correlated to s o i l pH. Of the s o i l elements, the two s o i l phosphorus analyses are p o s i t i v e l y correlated. S o i l copper co r r e l a t e s p o s i t i v e l y with s o i l zinc, s o i l pH and Mehlich 3 s o i l phosphorus, while Bray s o i l phosphorus cor r e l a t e s negatively to s o i l pH. There i s a p o s i t i v e c o r r e l a t i o n of colonization to s o i l zinc, and negative ones to f o l i a r phosphorus, f o l i a r zinc, f o l i a r copper, and to both methods of s o i l phosphorus analysis. Colonization i s not s i g n i f i c a n t l y correlated to s o i l copper or pH. 44 I§ble XVI: Correlation matrix for the data from Chapter Two (Pearson pairwise co r r e l a t i o n s ) . FolP* FolZn EQlCu SoilPM SoilZn SoilCu SoilPB FolP 1. 00 FolZn 0. 52 1. 00 FolCu 0. 85 0. 67 1. 00 SoilPM NS NS NS 1. 00 SoilZn -0. 33 NS NS NS 1. 00 SoilCu NS NS NS 0. 53 0. 28 1. 00 SoilPB 0. 23 NS NS 0. 79 NS NS 1. 00 Col -0. 29 -0. 39 -0. 37 -0. 36 0. 24 NS -0. 31 SoilpH 0. 42 NS 0. 42 NS NS 0. 25 -0. 29 1.00 NS 1.00 •FolP = f o l i a r phosphorus; FolZn = f o l i a r zinc; FolCu = f o l i a r copper; S o i l PM = s o i l phosphorus (Mehlich 3); SoilZn = s o i l zinc; SoilCu = s o i l copper; SoilPB = s o i l phosphorus (Bray PI); Col = percent colonization; SoilpH = s o i l pH; NS = not s i g n i f i c a n t at p < 0.05. QeiQQizstioQ Patterns The trends i n colonization observed at Oyster River and Agassiz (Fig. 7) were s i m i l a r . t o those reported by Dodd and J e f f r i e s (1986), Buwalda et a l . (1985b) and Yocum et a l . (1985), i n which mycorrhizae were present at low l e v e l s i n the f a l l and early spring, increased to a peak at anthesis, and then decreased again by harvest. Interestingly, the delayed colonization seen at Delta and Chilliwack has also been reported from other parts of the world (Hayman 1970, Jakobsen and Nielsen 1983, Hetrick and Bloom 1983, Hetrick et a l . 1984). A lack of f a l l c olonization i n the past has been att r i b u t e d i n part to a lack of sampling i n the f a l l or to inaccurate assessment methods (Dodd and J e f f r i e s 1986). However, t h i s does not apply to the s i t e s used i n t h i s study. The general patterns observed at a l l four s i t e s from May onward were most l i k e l y d i r e c t l y influenced by the behaviour of the host plants, although colonization i n the spring at Delta may have been affected by the grazing that the s i t e received i n December (Bethlenfalvay et a l . 1985). In the spring, the growth rates of both the fungus and the wheat roots would be expected to increase gradually. Because colonization i s determined on a f r a c t i o n a l , or percent of root basis, for increases i n colonization to occur the rate of spread of the mycorrhizal fungus must be greater than that of the roots (Dodd and J e f f r i e s 1986, Buwalda et a l . 1985b). This was apparently true at the Oyster River, Agassiz and Delta s i t e s . At anthesis i n June, 46 however, the roots of winter wheat plants stop growing and plant energy i s devoted to grain production (Barraclough 1984). If the fungus continues to spread at i t s previous rate, a sharp increase i n percent colonization w i l l occur, as was observed at a l l four s i t e s . As the grain ripens, photosynthesis slows and nutrients are translocated from the leaves to the grain (Karlen and Whitney 1980). The l e v e l of photosynthate sent to the roots declines, causing the endophyte to change from the active arbuscular state to the r e s t i n g v e s i c u l a r state. This would account f o r the decline in c o l onization observed at a l l s i t e s i n August. Although host physiology explains to some extent the general patterns of colonization, there does not appear to be a simple reason f o r the absence of colonization i n the f a l l at Chilliwack and Delta. As was mentioned i n the introduction to t h i s chapter, several explanations have been proposed. The r e s u l t s from t h i s study w i l l now be discussed with regard to these theories. Temperature Cool f a l l temperatures are considered by a number of researchers to delay colonization (Daniels and Trappe 1980, Hetrick and Bloom 1984, Hetrick et a l . 1984), but t h i s does not appear to be the delaying factor at the s i t e s used i n t h i s study (Tables IV and V). In fact, the coolest of the s i t e s . Oyster River, was colonized i n the f a l l , and Chilliwack, the warmest s i t e , was not. This i s best demonstrated i n Figure 8, where colonization was plotted 47 against, thermal time. If temperature had been the l i m i t i n g factor, a thermal time plot would have produced s i m i l a r patterns f o r a l l of the s i t e s , rather than emphasizing the differences, as t h i s one does. The normal s o i l temperatures i n the region are also s l i g h t l y colder than those reported i n Kansas by Hetrick et a l . (1984), who are among the strongest proponents of the temperature theory. Temperature may be a more important factor i n the winter, delaying possible spring colonization. Inoculum Density The second theory proposed to explain a lack of f a l l c o lonization implies that inoculum density i s too low to allow colonization to be established before winter (Jakobsen and Nielsen 1983, Hetrick and Bloom 1984). Unfortunately, t h i s theory cannot be addressed i n t h i s study, as no assessments were made of inoculum potential. Spores were found at a l l of the s i t e s i n the December s o i l samples (Table VII), however, in d i c a t i n g that inoculum was present at a l l s i t e s i n the f a l l . The inoculum density theory may have some merit, though, as Ianson and Linderman (1987) have demonstrated with pot studies that l o c a l i z i n g inoculum s i g n i f i c a n t l y increases early colonization i n winter wheat. Physical and Chemical Properties S o i l pH at these s i t e s ranged from about 5.6 to 4.1 (Fig. 16), which i s s i m i l a r to others reported (Jakobsen and Nielsen 1983, Young et a l . 1985). Although there was no s i g n i f i c a n t c o r r e l a t i o n of pH to colonization i n t h i s study, 48 pH has been shown to a f f e c t spore germination (Daniels and Trappe 1980, Abbott and Robson 1985, Killham 1985). Young et a l (1985) considered pH to have the strongest influence i n determining which species dominated i n winter wheat f i e l d s . As the pH at Chilliwack was consistently lower than those at the other s i t e s , i t may be an influencing factor there. However, since Delta had the highest pH of the s i t e s , i t may be a lesser factor at that s i t e . S o i l moisture can reduce spore germination (Daniels and Trappe 1980, S y l v i a and Schenck 1983) and spore abundance (Anderson et a l . 1984) under conditions of too much or too l i t t l e water. In winter wheat, mycorrhizae are reported to improve resistance to drought stres s (Allen and Boosalis 1983), and i n f e c t i o n tends to be stimulated i n plants growing i n droughty conditions (Dodd and J e f f r i e s 1986). High water, on the other hand, has been known to reduce i n f e c t i o n i n plants which are highly mycorrhizal i n d r i e r s o i l s . Anderson et a l . (1984) observed that three grass species with functional mycorrhizal associations i n dry s i t e s with low nutrient l e v e l s lacked functional mycorrhizae at wet s i t e s with high nutrient , l e v e l s , and Redhead (1975) reports s i m i l a r r e s u l t s . S o i l moisture l e v e l s d i f f e r e d considerably among the s i t e s . Chilliwack, which was poorly drained, received the most r a i n f a l l ; Delta received the l e a s t (Table IV). Oyster River, with i t s well-drained sandy s o i l , occasionally experienced drought. As most studies of winter wheat did not report moisture or r a i n f a l l r e s u l t s , i t i s d i f f i c u l t to compare those of t h i s 49 study to them. However, i t would appear that, possibly, the dry s o i l conditions at Oyster River stimulated colonization, while the wet conditions at Chilliwack i n h i b i t e d i t . None of the plants at the four s i t e s used i n t h i s study exhibited nutrient deficiency symptoms, and the s o i l analysis r e s u l t s indicate that they contained adequate to high l e v e l s of phosphorus <Figs. 12 and 13), zinc <Fig. 14) and copper (Fig. 15), except Agassiz, which was a b i t low i n zinc. The f o l i a r r e s u l t s are also within the expected ranges for winter wheat (Karlen and Whitney 1980) at the f i v e growth stages at which samples were taken. Of p a r t i c u l a r i n t e r e s t are the s o i l phosphorus r e s u l t s (Figs. 12 and 13), as the Chilliwack and Delta s i t e s had much higher l e v e l s than did the Oyster River and Agassiz s i t e s . High s o i l phosphorus l e v e l s have almost invariably been shown to reduce mycorrhizae (Sparling and Tinker 1978, Daniels and Trappe 1980, Graham et a l . 1981, Buwalda et a l . 1985a) by decreasing the root exudation which i s needed f o r root i n f e c t i o n to occur. Khan (1975) also reports that high phosphorus l e v e l s reduce spore formation. As with moisture, i t i s d i f f i c u l t to compare the r e s u l t s from t h i s study d i r e c t l y with those of other,winter wheat studies because s o i l nutrient l e v e l s have not been consistently reported. However, i t does appear that the very high s o i l phosphorus l e v e l s at Chilliwack and Delta may have i n h i b i t e d mycorrhizal development i n the f a l l , although colonization was established i n the spring without a s i g n i f i c a n t reduction i n phosphorus l e v e l . Graham et a l . (1981) suggest 50 that, t h i s may be due to increasing s o i l temperatures, which d i r e c t l y a l t e r the permeability of the root membrane to increase exudation without a f f e c t i n g phosphorus n u t r i t i o n . That phosphorus may be an i n h i b i t i n g factor i s supported by the negative c o r r e l a t i o n of s o i l phosphorus to colonization (Table XIV). The micronutrients do not appear to influence c o l o n i z a t i o n on these s i t e s . Host C u l t i v a r and/or Fungal Species The host c u l t i v a r i s unl i k e l y to be an influence on colonization i n t h i s study, as the same c u l t i v a r was used at a l l four s i t e s . It may however a f f e c t comparison to other studies. Fungal species i s a more l i k e l y influence, as t h i s was highly variable among the s i t e s (Table XII). Only a few other studies l i s t the fungal species used or found at the s i t e s (Young et a l . 1985, Hetrick et a l . 1984), and these are quite d i f f e r e n t from those here i n B r i t i s h Columbia. Young et a l . (1985) found that species d i f f e r e d i n pH and nutrient l e v e l optimums, and si m i l a r r e s u l t s have been reported elsewhere with respect to these factors and others such as moisture (Sparling and Tinker 1978, S i l v i a and Schenck 1983, Abbott and Robson 1985). No conclusions can be made about the factors a f f e c t i n g the i n d i v i d u a l species found from the s i t e s i n t h i s study, however, without further investigation, as the spores found may not be i n d i c a t i v e of the fungal species i n f e c t i n g the roots. Nonsporulating species may also have been present (Koske 1987). The observation of both coarse and f i n e endophyte 51 has been reported previously i n winter wheat (Dodd and J e f f r i e s 1986, Buwalda et a l . 1985b, Jakobsen and Nielsen 1983), but was not examined to any degree i n t h i s p a r t i c u l a r study. However, they are believed to behave in a s i m i l a r way with respect to i n f e c t i o n l e v e l and progress (Buwalda et a l . 1985a). MaQ!9<i!D§JQt Practices The past cropping history and the drainage were the only variables i n the management of these s i t e s , and either might e f f e c t the development of colonization. Drainage and i t s r e l a t i o n to s o i l moisture has already been discussed. The past cropping history i s important, as fallow periods have been shown to decrease the incidence of mycorrhizal fungi (Yocum et a l . 1985). In addition, d i f f e r e n t host species may s e l e c t for d i f f e r e n t species of mycorrhizal fungi. From the r e s u l t s of t h i s study, i t i s not possible to determine i f management practices influenced the observed colo n i z a t i o n patterns. S i g n i f i c a n c e to Crop Due to a lack of s u i t a b l e controls (free of VA mycorrhizae) at the four s i t e s used i n t h i s study, i t i s d i f f i c u l t to assess the importance of colonization to winter wheat i n South Coastal B r i t i s h Columbia. The r e s u l t s indicate that at high l e v e l s of s o i l nutrients plants with low l e v e l s of colonization have s i m i l a r l e v e l s of f o l i a r nutrients to plants with high l e v e l s of colonization, which concurs with the r e s u l t s of Menge (1983) and Plenchette et 52 a l . <1983). The v a r i e t i e s of winter wheat i n use have been bred for a maximum growth response to f e r t i l i z e r s and a maximum resistance to disease organisms such as fungi. From t h i s study one cannot say that the plants which are colonized are be n e f i t t i n g from the symbiosis, p a r t i c u l a r l y i n terms of nutrient uptake, as a l l of the s i t e s had more than adequate nutrient l e v e l s . Other, l e s s obvious benefits, however, may occur through increased water uptake and disease resistance, and these may ultimately be more important benefits, i n f i n a n c i a l terms, to wheat farmers i n t h i s region. Further studies appear to be necessary i n view of t h i s . 53 The r e s u l t s of t h i s study indicate that the colonization patterns of VA mycorrhizae i n winter wheat can be highly variable, even within a region such as South Coastal B r i t i s h Columbia. Host physiology i s a major determinant of the l e v e l s of colonization, but external environmental fac t o r s are apparently also influences. The most important of these appear to be moisture and s o i l phosphorus l e v e l , followed by fungal species, pH, and management practices. Temperature appears to have l i t t l e e f f e c t here i n B r i t i s h Columbia. Inoculum density and host c u l t i v a r may also be important, but t h i s cannot be determined from the r e s u l t s of t h i s study. Further inves t i g a t i o n i s necessary to e s t a b l i s h the benefits, i f any, winter wheat plants gain from the mycorrhizal symbiosis. It would appear that high s o i l nutrient l e v e l s can e a s i l y compensate for any n u t r i t i o n a l gains which an endophyte might provide, but there may be important benefits, such as increased water uptake and disease resistance, which were not apparent from t h i s study. This research also demonstrates the need for some consistency i n evaluating and reporting environmental fact o r s such as s o i l physical and chemical information i n studies of VA mycorrhizae, p a r t i c u l a r l y those involving f i e l d work. 54 CHAPTER THREE THE EFFECTS OF FOLIAR SPRAYS OF TWO SYSTEMIC FUNGICIDES ON VA MYCORRHIZAE IN WINTER WHEAT. l O t r - O d u c t i g n The use of pesticides to control organisms which may-damage crop plants i s a well-established practice i n modern agriculture. The elimination of pathogens leads to improved plant health, which i n turn r e s u l t s i n an increased y i e l d . Pesticides may be used for the prevention of disease by i n h i b i t i n g pathogens before the host becomes infected, or to cure disease by eradicating established pathogens (Wain and Carter 1977a). Although substances have been applied onto plants f o r many centuries, the research and development of modern fungicides to control plant disease began i n the early 1900's (Wain and Carter 1977b). Generally, a fungicide i s an agent that k i l l s fungi. A fungicide which i n h i b i t s fungi i n some way without k i l l i n g i s a fungistat. There are two main groups of fungicides (Wain and Carter 1977a). Systemic fucgioicies are absorbed by the plant and are translocated to regions away from the s i t e of application, while ngnsystemic or contact fungicides are l o c a l i z e d i n the area to which they are applied. These chemicals may be used as s o i l drenches, seed treatments, or as f o l i a r sprays. The widespread use of fungicides i n a g r i c u l t u r e has, i n the l a s t two decades, directed attention toward the p o t e n t i a l t o x i c i t y of these chemicals to nontarget fungi 55 such as those involved i n VA mycorrhizae. Although any improvements i n the health of the host plant would t h e o r e t i c a l l y benefit mycorrhizal fungi, some of the chemicals used may be harmful to these endosymblonts, either v i a d i r e c t t o x i c i t y , or i n d i r e c t l y through changes which the fungicide may Induce i n the physiology of the host plant. There have been a number of studies i n v e s t i g a t i n g the e f f e c t s of fungicides on VA mycorrhizae, focussing on only a few of the many availa b l e compounds. The r e s u l t s are varied, and appear to depend on the p a r t i c u l a r fungicide used and the way i n which i t i s applied to the host plant. Generally, contact fungicides are le s s t o x i c to VA mycorrhizae than are systemic ones (Nemec I960, Menge 1982, Vyas 1988). Of the application methods, s o i l drenches are most deleterious. Benomyl, which i s systemic, has been shown to severely reduce mycorrhizal i n f e c t i o n when applied to the s o i l (Sutton and Sheppard 1976, Kough et a l . 1977, Bailey and S a f i r 1978, Boatman et a l . 1978, Williams and Beane 1982, Spokes et a l . 1982, Hale and Sanders 1982, Carr and Hinkley 1985, F i t t e r 1986, F i t t e r and Nichols 1988) and can reduce phosphorus uptake i n the host plant (Boatman et a l . 1978, Hale and Sanders 1982, F i t t e r and Nichols 1988). Other systemics, including iprodione and thiram (Spokes et a l . 1982), thiophanate-methyl (Boatman et a l . 1978), thiabendazole and quintozen (Sutton and Sheppard 1976) w i l l also i n h i b i t VA mycorrhizae when applied to s o i l . However, neither the systemic metalaxyl (Groth and Martinson 1983) nor the contact fungicide captan (Tommerup and Briggs 1981, 56 Sutton and Sheppard 1976) as s o i l treatments affected mycorrhizae i n any way. Seed treatments with a variety of both systemic and contact fungicides w i l l also i n h i b i t VAM <Jalali and Domsch 1975, Mott and Odvody 1987), but not as severely as when used as s o i l treatments (Spokes et a l . 1982). The e f f e c t s of seed treatments usually d i s s i p a t e quickly (Smith 1978). F o l i a r fungicide treatments have produced the widest range of r e s u l t s . J a l a l i and Domsch (1975) and Rhodes and Larsen (1981) report that f o l i a r a p p l i c a t i o n of some systemic and contact fungicides r e s t r i c t e d mycorrhizal development. In contrast the systemic maneb was found to have no e f f e c t on colonization l e v e l s and spore production (Nemec 1980) while f o s e t y l - A l , also a systemic fungicide, stimulated root colonization <Jabaji-Hare and Kendrick 1985). Dehne (1987) also reports p o s i t i v e influences of f o l i a r fungicide treatments on VA mycorrhizae. In view of these c o n f l i c t i n g reports, i t i s d i f f i c u l t to generalize about, or to predict, the e f f e c t s of f o l i a r fungicide treatments on VA mycorrhizae. As well, v i r t u a l l y a l l previous fungicide studies have been conducted i n greenhouses or growth chambers, and may not adequately represent the reactions of mycorrhizae i n f i e l d crops. Thus, the objective of t h i s study i s to monitor the e f f e c t s of two s i m i l a r systemic fungicides, applied f o l i a r l y , on VA mycorrhizae i n field-grown winter wheat i n South Coastal B r i t i s h Columbia, i n view of changes i n colonization patterns and f o l i a r nutrient content over a 20 day period 57 a f t e r spraying. 58 Materials and Methods Ei£l^ S i t e This study was conducted at the University Of B r i t i s h Columbia Oyster River Research Farm. A more detailed description of the s i t e can be found i n Chapter Two (Figure 1 and Table I>. This s i t e was selected for t h i s study because i t was colonized by VA mycorrhizae at the time of the f i r s t fungicide application, and because i t had a low incidence of disease. The plots used (Figure 20) were part of a larger study in v e s t i g a t i n g the e f f e c t s of plant growth regulators and fungicides i n varying combinations (Temple and Bomke 1989). For t h i s p a r t i c u l a r study, only two of the si x 9.0m x 10.0m plots i n each of the four r e p l i c a t e randomized blocks were used. One plot was a control, which did not receive any treatments, and one was a treated plot, which received both fungicides. Fungicides R The fungicides used i n t h i s study were Bayleton (50% R triademifon as a wettable powder) and T i l t (250 g/L propiconazole as an emulsifiable concentrate). Bayleton i s manufactured by Chemagro Ltd, and has the chemical structure: 1-(4-chlorophenoxy) 3,3-dimethyl-l-(1, 2, 4-triazol-l-yl)butan-2-one. It i s used to control rust and powdery mildew, and i n t e r f e r e s with membrane synthesis and function by interrupting ergosterol biosynthesis (Vyas 1988). T i l t i s produced by Ciba-Geigy Canada Ltd., and i t s chemical structure i s : 1-(2-(2,4-dichlorophenyl)-4-propyl-59 Figure 20: A photograph of the study area used f o r the fungicide t r i a l s , at the time of Bayleton application. 1,3-dioxolan-2-yl methyl)1H-1,2,4-triazole. It controls powdery mildew and septoria via the same metabolic pathway as Bayleton (Vyas 1988). Both of these chemicals function as both contact and systemic fungicides, and are believed to be translocated acropetally i n the apoplast (Scheinpflug and Kuck 1987). They are e f f e c t i v e against a broad spectrum of Ascomycetes, Basidiomycetes and Fungi Imperfect!. In t h i s study, both were applied at the recommended dosage rates, using a mechanical sprayer towed behind a t r a c t o r . Sampling Methods Samples for both fungicide t r i a l s were taken every four days, beginning with the date on which the fungicide was sprayed (Table XVII). Representative pictures of the growth stages of the plants at the two sampling dates are shown i n Figure 21. Destructive samples were taken for the Bayleton 60 Table XVII: Sampling dates for the fungicide t r i a l s (1988). Sampling Date Day Bayleton T i l t 0* A p r i l 8 June 6 4 A p r i l 12 June 10 8 A p r i l 16 June 14 12 A p r i l 20 June 18 16 A p r i l 24 June 22 20 A p r i l 28 June 26 Growth Stage 25-31 50-70 •The f i r s t samples were taken on the day • that the fungicide was sprayed; growth stages were determined using the Zadok i scale (Tottman and Makepeace 1979). i t r i a l i n a s i m i l a r manner to the early samples of Chapter Two (Figure 5a), while the sampling method f o r the T i l t harvest was s i m i l a r to that.used for the l a s t two harvests i n Chapter Two (Figures 5b,5c). For both fungicide t r i a l s , 10 samples were taken from each plot per block per date, and were bulked together. The f o l i a r and root samples were preserved and analysed as i n Chapter Two; the s o i l samples were discarded. The s t a t i s t i c a l analyses were also done as was described i n Chapter Two, with log (5il + 1) transformations done to the percent colonization (%I> re s u l t s . 61 Photographs of the growth stages of the plants when the fungicides were applied. [<a> Bayleton t r i a l , GS 25-31; <b) T i l t t r i a l , GS 50-70.] 62 Results Bayleton T r i a l Figure 22 displays the e f f e c t s of Bayleton on colonization. S t a t i s t i c a l l y , treatment, date and treatment* date were s i g n i f i c a n t at p < 0.05 (Table XVIIIa). For the f i r s t 8 days afte r spraying, there were no s i g n i f i c a n t differences between colonization patterns on the treated and the control plots (Table XVIIIb). However, on day 12, colonization dropped sharply on the sprayed plots as compared with the controls. The l e v e l increased again by day IS, and by day 20 were within the same range as those on the control plots (4-6%). . Levels of f o l i a r phosphorus (Figure 23) and zinc (Figure 24) both generally decreased over time, and both elements were s l i g h t l y higher i n the treated plots. Treatment, date and treatment*date i n t e r a c t i o n s were s t a t i s t i c a l l y s i g n i f i c a n t for both elements (Tables XlXa and XXa). Interestingly, the differences i n f o l i a r nutrients between the treatments decreased a f t e r day 12 (Tables XlXb and XXb). The l e v e l s of f o l i a r copper were below 1 mg/kg for the treatment and control, and did not change s i g n i f i c a n t l y over the time period of t h i s t r i a l . Consequently, they were not included. 63 Table XVIIla: Analysis of variance for colonization (Bayleton fungicide). Sum of Mean Source Sguares BE Sguares F-ratig Probability Treatment 2. 06 1 2. 06 49. 65 0. 000 Date 2. 35 5 0. 47 11. 36 0. 000 Treatment* Date 1. 74 5 0. 35 8. 39 0. 000 Error 11. 42 276 0. 04 Total 17. 56 287 Table XVIIIb: Mean colonization i n the Bayleton fungicide study (percent). TREATMENT Date With Fungicide Without Fungicide Day 0 4 be* 6 c Day 4 5 be 4 be Day 8 4 be 6 c Day 12 l a 5 be Day 16 3 b 6 c Day 20 4 be 5 be » Those followed by different letters d i f f e r s i g n i f i c a nt ly using Tukey's (HSD) Test (p < 0.05). , S4 5 ho 3 o H — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i i i O 4 8 12 16 20 T i m e ( d a y s ) 23: E f f e c t s of Bayleton fungicide on f o l i a r phosphorus. Table XlXa: Analysis of variance table for f o l i a r phosphorus (Bayleton fungicide). Sum of Mean Source Sguares QF Sguares Probability. Treatment 4. 07 1 4. 07 92. 43 0. 000 Date 15. 51 5 3. 10 70. 53 0. 000 Treatment* Date 0. 82 5 0. 16 3. 71 0. 008 Error 1. 58 38 0. 04 Total 21. 98 47 Table XlXb: Mean f o l i a r phosphorus i n the Bayleton fungicide study (g/kg). TREATMENT Date With Fungicide Without Fungicide Day 0 4.20f* 3.12de Day 4 3. 35e 2.62bcd Day 8 2.69cd 2.12ab Day 12 2.38abc 2. 08a Day 16 2.31ebc 1. 91a Day 20 2.29abc 1. 88a i * Those followed by different letters d i f f e r significantly using Tukey's (HSD) Test (p < 0.05). 65 40 feO bj 30 -o - Treated Plots O-C o n t r o l Plots 0 -\ 1 1 1 1 1 1 r 0 4 ~ i 1 1 1 1 1 1 1 i i i r — 8 12 16 20 Time (days) Eigyre 24: E f f e c t s of Bayleton fungicide on f o l i a r zinc. I i e i e XX.a: Analysis of variance table f o r f o l i a r zinc (Bayleton fungicide). Sum of Mean Source Sguares BE Sguares F-ratio Probability Treatment 308.92 1 308.92 70. 16 0. 000 Date 766.09 5 153.22 34. 80 0. 000 Treatment* Date 87. 91 5 17. 58 3. 99 0. 006 Error 158.51 36 4. 40 Total 1321.40 47 Table XXb: Mean f o l i a r zinc i n the Bayleton fungicide study (mg/kg). TREATMENT Date With Fungicide Without Fungicide Day 0 27.63d* 17.31bc Day 4 20.98c 14.19ab Day 8 14.89ab 10.93a Day 12 13.39ab 10.99a Day 16 13.66ab 10.24a Day 20 13.31ab 9.76a » Those followed by different letters d i f f e r s i g n i f i c a n tl y using Tukey's (HSD) Test (p < 0.05). 66 T i l t T r i a l The colonization patterns found i n the t r i a l with the fungicide T i l t are shown i n Figure 25. The percent colonization l e v e l s were generally higher than those found during the Bayleton t r i a l (about 8 versus about 5), which i s most l i k e l y due to differences i n times of sampling, as i s explained i n Chapter Two. The patterns of t h i s t r i a l d i f f e r considerably from those following the application of Bayleton. S t a t i s t i c a l l y , treatment and date were s i g n i f i c a n t at p < 0.05, but there was no s i g n i f i c a n t treatment»date i n t e r a c t i o n (Table XXIa). This indicates that the treated and control plots did not d i f f e r s i g n i f i c a n t l y i n t h e i r l e v e l s of colonization over the 20 day period examined (Table XXIb). Spraying with T i l t did not s i g n i f i c a n t l y a f f e c t f o l i a r phosphorus either (Figure 26, Tables XXIIa, XXIIb), which ranged between 2.22 and 2.88 g/kg. With respect to f o l i a r zinc, the only time when the treated and control p l o t s were s i g n i f i c a n t l y d i f f e r e n t was on the day that the fungicide was applied (Figure 27, Tables XXIIIa, XXIIIb), when the l e v e l i n the treated plots soared to 57.22 mg/kg, compared with 23.96 i n the untreated plots on that date and a range of 16.58 to 27.17 mg/kg for the re s t of the observation period. This i s probably not a true difference, though, and i s most l i k e l y due to contamination from the fungicide or sprayer. The r e s u l t s from analysis f o r f o l i a r copper for t h i s fungicide t r i a l were s i m i l a r to those for the Bayleton t r i a l , and thus were also not included. 67 40 PI O ffl N -|H PI o I—I o u 30 -20 -• - Treated Plots O-Control Plots ~ i — 12 i — 16 20 Time (days) E i a y r e 25: E f f e c t s of T i l t fungicide on colonization. Table XXIa: Analysis of variance table for colonization ( T i l t fungicide). Sum of Mean Source Sguares DF Sguares Ezratlg Probability. Treatment. 0. 54 1 0. 54 8. 76 0. 003 Date 0. 77 5 0. 15 2. 48 0. 032 Treatment* Date 0. 50 5 0. 10 1. 61 0. 157 Error 17. 05 276 0. 06 Total 18. 86 287 Table XXIb: Mean colonization i n the T i l t fungicide study (percent). fiate With Fungicide Day 0 8 ab* Day 4 9 b Day 8 8 ab Day 12 9 b Day 16 8 ab Day 20 7 ab TREATMENT Without Fungicide 6 ab 5 ab 7 ab 8 ab 9 b 4 a Those followed by different letters d i f f e r significantly u B i n g Tukey's (HSD) Test (p < 0.05). 68 5 PH 1 -to o A T 1 ,-8 12 16 T i m e ( d a y s ) E i a a c e 26s E f f e c t s of T i l t fungicide on f o l i a r phosphorus. Table XXIla: Analysis of variance table for f o l i a r phosphorus ( T i l t fungicide). Sum of Mean Source Sguares BE Sguares F-ratig Probability Treatment 0. 016 1 0. 016 0. 67 0. 418 Date 1. 55 5 0. 31 12. 83 0. 000 Treatment* Date 0. 15 5 0. 03 1. 25 0. 308 Error 0. 85 35 0. 24 Total 2. 59 46 Table XXIlb: Mean f o l i a r phosphorus i n the T i l t fungicide study (g/kg). TREATMENT Date With Fungicide Without Fungicide Day 0 2.77bc* 2. 88c Day 4 2.44ab 2. 38a Day 8 2. 24a 2. 27a Day 12 2. 32a 2. 22a Day 16 2.50abe 2. 26a Day 20 2. 31a 2. 35a » Those followed by different letters d i f f e r significantly using Tukey's (HSD) Test <p < 0.05). 69 60 fl io -N 0 H 1 1 1—i 1—i 1—i 1 1—i 1 1 1 — i 1—i 1 1 — 0 4 8 12 16 20 Time (days) EiQMHe 27: E f f e c t s of T i l t fungicide on f o l i a r zinc. Table XXIIIa: Analysis of variance table for f o l i a r z i nc ( T i l t fungicide). Sum of Mean Source Sguares BE Sguares F-ratio Probability. Treatment 630. 5 1 630.54 6. 97 0. 012 Date 3157.5 5 631.50 3. 08 0. 000 Treatment* Date 1390.1 5 278.01 3. 08 0. 021 Error 3164.3 35 90. 41 Total 8492.4 46 Table XXIIlb: Mean f o l i a r zinc i n the T i l t fungicide study (mg/kg). .. - • -TREATMENT Bate With Fungicide Without Fungicide Day 0 57.22b» 23.96a Day 4 19.89a 18.07a Day 8 27.17a 20.43a Day 12 19.10a 16.58a Day 16 21.13a 18.57a Day 20 19.91a 19.08a * Those followed by different letters d i f f e r significantly using Tukey's (HSD) Test (p < 0.05). 70 Correlations Table XXIV indicates that there were no s i g n i f i c a n t c o r r e l a t i o n s of colonization to f o l i a r elements for either fungicide t r i a l , but that a l l of the f o l i a r elements were s i g n i f i c a n t l y correlated. Table XXIV: Correlation matrix f o r the Data from Chapter Three (Pearson pairwise c o r r e l a t i o n s ) . BCol * BFolP iEolZn TCol TEQlP TFolZn BCol 1. 00 BFolP NS 1. 00 BFolZn NS 0. 94 1. 00 TCol NS NS NS 1. 00 TFolP NS 0. 63 0. 54 NS 1. 00 TFolZn NS 0. 61 0. 49 NS 0.49 1.00 *B = Bayleton t r i a l ; T = T i l t t r i a l ; Col. = percent col o n i z a t i o n ; FolP = f o l i a r phosphorus; FolZn = f o l i a r z i n c ; NS = not s i g n i f i c a n t at p < 0.05. 71 Discussion Mycorrhizal colonization l e v e l s i n winter wheat i n the growth stage range of 25-31 on the Zadok scale dropped sharply within 12 days following f o l i a r a p p l i c a t i o n of the systemic fungicide Bayleton <triademifon), but increased again to the l e v e l s found i n the control plots by 20 days af t e r treatment. In contrast, there were no s i g n i f i c a n t d ifferences i n colonization between treated and control p l o t s over a 20 day period when T i l t , which i s also a systemic fungicide, was applied to plants i n the growth stage range of 50-70. Past studies with Bayleton also i n d i c a t e that f o l i a r a pplication can reduce VA mycorrhizae. J a l a l i and Domsch (1975) had reduced colonization when Bayleton was applied to three-week-old spring wheat plants, but unfortunately did not t e s t any other growth stages. Rhodes and Larsen (1981), working with creeping bentgrass (Agrgstis g a l u s t r i s Huds. > found that Bayleton reduced mycorrhizal development when i t was applied at 4-8 weeks after seeding, but had no detectable e f f e c t when applied 16-20 weeks a f t e r seeding. When applied to s o i l , the suppression of VA mycorrhizae i n onion plants by Bayleton was most apparent 5 weeks a f t e r treatment, but was, for the most part, overcome within 15 weeks (Spokes et a l . 1981). Thus, i t would appear that Bayleton can have a f u n g i s t a t i c e f f e c t on VA mycorrhizal fungi. Regretably, no studies examining the e f f e c t s of T i l t (propiconazole) on VA mycorrhizae have been published, and so no comparisons can 72 be made f o r the i n d i c a t i o n s of t h i s study that i t does not a f f e c t these endophytes. There are a number of possible explanations for the differences between the r e s u l t s from these two fungicides. The f i r s t centers on the differences i n chemical structures between the two chemicals. Past studies have shown that the e f f e c t s of d i f f e r e n t chemical groups on VA mycorrhizae can vary (Trappe et a l . 1984). However, the differences between Bayleton and T i l t are very s l i g h t i n terms of t h e i r behaviours and modes of action (Scheinpflug and Kuck 1987), so t h i s i s u n l i k e l y to explain e n t i r e l y t h e i r d i f f e r e n t e f f e c t s on colonization. A second, possible explanation i s that species of plants or fungi involved i n mycorrhizae may react i n d i f f e r e n t ways to the same fungicide, or to two very s i m i l a r fungicides. This theory has been examined by a number of researchers, and appears to be quite v a l i d (Smith 1978, Spokes et a l . 1981, Menge 1982, Trappe et a l . 1984). Variations i n the responses of d i f f e r e n t species of host plant, and subsequent e f f e c t s on mycorrhizal fungi, have not been examined i n previous studies, but could also be important. As the same wheat c u l t i v a r was used f o r both fungicide t r i a l s , the importance of host species i n producing these r e s u l t s i s s l i g h t . However, i t could account f o r any differences i n t h i s study from other studies. It i s not possible to t e l l whether fungal species had an influence on these r e s u l t s , as only s u p e r f i c i a l attempts were made to i d e n t i f y the fungi involved i n these mycorrhizae. Futher research i s warranted 73 i n -these areas. A more l i k e l y theory to explain the r e s u l t s of Bayleton and T i l t i n t h i s study concerns the growth stages of the plants when the fungicides were applied. When Bayleton was sprayed, stem elongation had not yet occurred. Consequently, the plants were small, and there were gaps i n the canopy (Figure 21a). Although translocation of both of these fungicides i s usually acropetal, very l i m i t e d basipetal transport has been observed (Smith 1976, Scheinpflug and Kuck 1987). In such small plants, translocation from the leaves to the roots could occur quite e a s i l y . As well, because these fungicides were applied by a mechanical sprayer which was towed behind a tractor, gaps i n the canopy would expose the s o i l to fungicide, and subsequently the plant roots and t h e i r endosymbionts. When T i l t was applied, the plants were much larger (Figure 21b> and translocation from the leaves to the roots would be l e s s l i k e l y . Moreover, the canopy was quite dense, and l i t t l e i f any fungicide could be sprayed onto the ground. Rhodes and Larsen (1981) also report differences i n the e f f e c t s of Bayleton at d i f f e r e n t times of application. Thus, the growth stage of the plants when the fungicide i s sprayed seems to be very important when the e f f e c t s of a fungicide on VA mycorrhizae was examined. One other theory proposed by many researchers to explain the e f f e c t s of systemic fungicides on VA mycorrhizae suggests that f o l i a r sprays of these chemicals may induce changes i n the metabolism of the host plant and thereby 74 i n d i r e c t l y disturb the endosymbiont ( J a l a l i and Domsch 1977, Smith 1978, Jabaji-Hare and Kendrick 1985, Dehne 1987, Schonbeck and Dehne 1987, Vyas 1988). The consequences of t h i s disturbance may have p o s i t i v e or negative implications f o r the mycorrhizal fungus. Both J a l a l i and Domsch (1977) and Jabaji-Hare and Kendrick (1985) observed that some systemic fungicides produced changes i n root exudations and amino acid metabolism. Different e f f e c t s of Bayleton and T i l t could account f o r the differences which these fungicides had on mycorrhizae i n winter wheat. These changes i n plant physiology could also vary with the growth stage of the host. However, as changes i n host metabolism were not monitored during these fungicide t r i a l s , t h i s cannot be considered a d e f i n i t e influence without further study. F i n a l l y , there may be a very simple explanation to account f o r the differences between the fungicides: sampling date. T i l t may have had a short-term e f f e c t which was missed by sampling every four days, rather than more frequently. A l t e r n a t i v e l y , the fungicide may have translocated very slowly to the roots to produce an e f f e c t some time aft e r the 20 day monitoring period. Again, further study i s required to support t h i s explanation. The s i g n i f i c a n c e to the wheat plants of the apparent disruption of the VA mycorrhizae which the Bayleton treatment caused i s d i f f i c u l t to ascertain. The nutrient s l e v e l s on t h i s s i t e were more than adequate (Chaper Two), and the colonization l e v e l s were not very high. The f o l i a r 75 analyses indicate that the treated plants had higher l e v e l s of phosphorus and zinc than the control plants, but t h i s difference became l e s s a f t e r day 12, which i s when colonization decreased. However, t h i s i s probably just a coincidence, because there were no s i g n i f i c a n t c o r r e l a t i o n s between the f o l i a r nutrients and colonization. The reason for the elevated nutrient l e v e l s on the treated plots i s unclear, but i s probably due to contamination from the fungicide, which would also account f o r the lessening of differences between the treatments over time. The application of T i l t does not appear to have had any e f f e c t on the winter wheat plants, as there were no differences i n f o l i a r nutrient l e v e l s on treated and control plots. It should be noted that the colonization l e v e l s observed i n A p r i l and June for t h i s study are lower than those reported i n Chapter Two for the same loc a t i o n (Oyster River). This i s mainly due to d i f f e r e n t sampling areas i n the f i e l d for the two studies: the study of paterns was conducted toward the centre of the f i e l d , while the fungicide t r i a l s were at the edge. Colonization tended to be more variable on t h i s outer edge than i t was i n the centre. 76 Conclusions The r e s u l t s of t h i s study indicate that the systemic fungicide Bayleton <triademifon) i s f u n g i s t a t i c on vesicular-arbuscular mycorrhizal fungi when applied at the Zadok growth stage range of 25-31 to plants i n the f i e l d . I t i s e f f e c t i v e within 12 days of spraying, but the e f f e c t does not appear to l a s t f or more than 20 days. Some elevation of f o l i a r phosphorus and zinc occurs i n the treated plants shortly a f t e r spraying, but t h i s i s probably due to contamination from the fungicide. T i l t <propiconazole), which i s also systemic, had no s i g n i f i c a n t e f f e c t on VA mycorrhizal and f o l i a r nutrient l e v e l s when i t was applied at the growth stage range of 50-70. Without further research, i t i s d i f f i c u l t to explain why these c l o s e l y related chemicals had such d i f f e r e n t e f f e c t s when used on the same plant species. The most l i k e l y explanations are: that the small differences i n chemical structure could r e s u l t i n large differences i n e f f e c t ; that the sampling dates used may have missed a reaction from the T i l t a p plication; and that the d i f f e r e n t growth stages of the plants when the fungicides were applied resulted i n altered behaviour of the chemicals. Other possible explanations include d i f f e r i n g e f f e c t s on the plant metabolism, and d i f f e r e n t fungal species when the fungicides were sprayed. '77 GENERAL SUMMARY AND CONCLUSIONS General Summary Varied r e s u l t s on patterns of VA mycorrhizal colonization from other parts of the world, and the factors a f f e c t i n g them, led to t h i s two-part i n v e s t i g a t i o n of VA mycorrhizae i n winter wheat i n South Coastal B r i t i s h Columbia. The f i r s t part of t h i s study was conducted at f i e l d s i t e s at Oyster River, Agassiz, Delta and Chilliwack. These f i e l d s i t e s were known to d i f f e r i n s o i l type, drainage and r a i n f a l l , and had f a i r l y s i m i l a r temperatures through the year. F o l i a r , s o i l and root samples were c o l l e c t e d at f i v e times during the growing season: December, February, May, June and August, using four r e p l i c a t e blocks per s i t e per harvest. Analyses were done for percent colonization of roots, f o l i a r phosphorus, zinc and copper, s o i l pH, s o i l phosphorus, zinc and copper. S o i l phosphorus was analysed by two d i f f e r e n t methods (Bray PI and Mehlich 3). F o l i a r samples were not c o l l e c t e d or analysed f o r the Delta s i t e i n February as i t had l i t t l e above-ground biomass due to heavy grazing i n December by snow geese. The Oyster River and Agassiz s i t e s were found to be colonized throughout the growing season, and the Oyster River plants were the most highly colonized of the plants at the s i t e s f o r a l l but the f i n a l harvest. Colonization at Chilliwack and Delta was not r e a l l y established u n t i l a f t e r May. A l l s i t e s showed peaks i n colonization l e v e l s i n June. 78 Early colonization was sparse i n root c e l l s , and was almost e n t i r e l y arbuscular. It increased to become quite dense i n June, with both v e s i c l e s and arbuscules. By the f i n a l harvest, though, i t was mainly vesicular. Both coarse and f i n e endophyte were present at a l l s i t e s . The fungal species varied among the s i t e s , and generally there were spores of more species present i n August than i n December. Both methods of s o i l phosphorus analysis indicated that Chilliwack and Delta had much higher l e v e l s of av a i l a b l e r s o i l phosphorus than did Oyster River and Agassiz. Oyster River had the highest s o i l z inc l e v e l s , while Delta had the most s o i l copper. The Agassiz s i t e had the lowest l e v e l s for both of these micronutrients. Generally, a l l s o i l nutrient l e v e l s were adequate f o r plant growth. A l l s i t e s were s l i g h t l y a c i d i c i n pH, with the lowest readings at Chilliwack. F o l i a r phosphorus, copper and zinc were about the same at a l l of the s i t e s , and generally decreased as the plants aged. The colonization patterns observed at these s i t e s were probably d i r e c t l y influenced by the behaviour of the host plants. Although cool f a l l temperatures are considered responsible f o r a lack of f a l l colonization i n other parts of the world, that i s not the case here, because Oyster River, which experienced the coolest f a l l temperatures, was colonized shortly a f t e r germination. Inoculation density may have been a factor, along with host c u l t i v a r and fungal species, but these require further study. The physical and 79 chemical properties of the s o i l were more l i k e l y influences on colonization, e s p e c i a l l y s o i l moisture and phosphorus. Management practices such as past cropping history and drainage may also be important, but require further investigation. In the second part of t h i s study, the regular use of systemic fungicides to control f o l i a r disease triggered an i n t e r e s t i n the e f f e c t s of such chemicals on VA mycorrhizal fungi. In a study conducted at the U. B. C. Oyster River Research Farm, two systemic fungicides were f o l i a r l y applied to winter wheat plants. These were: Bayleton (triademifon) at the Zadok growth range of 25-31, and T i l t (propiconazole) i n the growth stage range of 50-70. Beginning on the date of spraying, f o l i a r and root samples were c o l l e c t e d every four days for a 20 day period. Analyses for percent colonization of roots and f o r f o l i a r phosphorus, zinc and copper were then conducted. Bayleton caused colonization to drop between days 12 and 20 i n the treated plants, but increased the l e v e l s of f o l i a r phosphorus and zinc. Copper did not s i g n i f i c a n t l y change. T i l t did not produce any s i g n i f i c a n t e f f e c t s with regard to colonization and f o l i a r elements. These very d i f f e r e n t r e s u l t s are most l i k e l y due to differences i n plant age when the chemicals were applied and to s l i g h t differences i n chemical structure. Other explanations, which require further study to substantiate, include p o t e n t i a l l y missed r e s u l t s due to the sampling times 80 chosen, d i f f e r e n t fungal species with d i f f e r e n t reactions to these chemicals, and d i f f e r i n g e f f e c t s of these fungicides on plant metabolism. From t h i s study, i t i s d i f f i c u l t to assess the importance of VA mycorrhizae to winter wheat plants i n South Coastal B r i t i s h Columbia. However, because s o i l nutrients are at more than adequate l e v e l s , they may not be very important n u t r i t i o n a l l y to these plants. This i s supported by the generally low colonization le v e l s , the s i m i l a r i t y i n f o l i a r nutrient l e v e l s regardless of colonization at the s i t e s , and the lack of e f f e c t on f o l i a r nutrients due to the short-term drop i n colonization caused by Bayleton. However, t h i s association may provide enormous benefits to the host plants i n ways such as drought tolerance and disease resistance which are not apparent from t h i s study. 81 General Conclusions 1. Winter wheat plants are colonized by VA mycorrhizae i n South Coastal B r i t i s h Columbia to varying extents. Colonization may or may not be established i n the f a l l . 2. Temperature does not strongly a f f e c t f a l l c olonization i n t h i s region. 3. S o i l moisture and phosphorus lev e l s , along with plant physiology, are the strongest influences on colonization i n winter wheat plants i n t h i s region. 4. 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In: Marsh, R. W., ed. Systemic Fungicides. Second ed i t i o n . Longman. New York. Wang, G. M., D. P. St r i b l e y , P. B. Tinker and C. Walker. 1985. S o i l pH and vesicular-arbuscular mycorrhizas. pp. 219-224. In: F i t t e r , A. H., ed. Ecologi c a l Interactions i n S o i l s : Plants, Microbes and Animals. Blackwell S c i e n t i f i c Publications. Oxford. Watanabe, F. S, and S. R. Olsen. 1965. Test of an ascorbic acid method for determining phosphorus i n water and NaHCO extracts from s o i l . S o i l S c i . Soc. Am. Proc. 29: 677-678. Williams, T. D., and J. Beane. 1982. E f f e c t s of aldicarb and benomyl on forage maize. pp. 158-159. In: Rep. Rothamsted Expt. Stn. f o r 1982. Yocum, D. H., H. J. Larsen, and M. G. Boosalis. 1985. The ef f e c t s of t i l l a g e treatments and a fallow season on V. A. mycorrhizae of winter wheat. p. 297. In: Molina, R., ed. Proc. 6th NACOM. For. Res. Lab., C o r v a l l i s , Oregon. Young, J. L., E. A. Davis and S. L. Rose. 1985. Endomycorrhizal fungi i n breeder wheats and t r i t i c a l e c u l t i v a r s field-grown on f e r t i l e s o i l s . Agron. J. 77: 219-224. 90 APPENDIX I RESULTS OF PATTERNS STUDY A) S o i l and F o l i a r Data OYSTER RIVER SOILPM SOILZn SOILCu SOILPB SOILpH LEAFP LEAFZn LEAFCu DEC 29. 45 1. 59 1. 62 61. 9 4. 9 2. 46 23. 78 3. 84 DEC 48. 30 2. 15 2. 63 84. 4 5. 0 3. 06 16. 59 4. 21 DEC 33. 78 1. 66 3. 04 59. 4 5. 3 2. 88 16. 16 3. 79 DEC 96. 52 2. 77 2. 77 106. 3 5. 1 3. 32 19. 56 4. 42 FEB 48. 32 2. 73 2. 46 88. 8 5. 1 2. 63 15. 98 4. 84 FEB 47. 84 3. 15 2. 95 70. 0 5. 2 2. 57 14. 23 4. 42 FEB 43. 5 2. 06 3. 22 59. 4 5. 1 2. 25 12. 38 3. 10 FEB 40. 76 2. 31 3. 42 63. 2 5. 2 2. 02 10. 83 2. 63 MAY 38. 31 2. 43 1. 98 69. 4 5. 1 1. 38 6. 54 1. 0 0 MAY 39. 04 2. 04 1. 97 67. 5 5. 0 1. 71 8. 87 1. 93 MAY 36. 25 1. 98 2. 81 56. 3 5. 1 1. 45 6. 79 1. 46 MAY 107. 50 3. 74 3. 75 97. 6 5. 0 1. 5 5 7. 29 1. 14 JUN 49. 99 2. 58 2. 66 78. 8 4. 6 1. 14 6. 18 1. 0 0 JUN 43. 57 2. 64 2. 60 89. 4 4. 8 1. 33 7. 41 1. 0 0 JUN 40. 54 2. 38 3. 72 68. 8 4. 8 1. 22 5. 71 1. 0 0 JUN 80. 59 2. 93 3. 52 108. 2 4. 9 1. 43 6. 31 1. 00 AUG 39. 73 2. 51 2. 26 66. 5 4. 7 0. 48 7. 00 1. 0 0 AUG 46. 67 1. 93 2. 55 92. 0 4. 8 0. 48 5. 21 1. 0 0 AUG 29. 72 2. 02 3. 11 46. 5 4. 8 0. 25 4. 13 1. 0 0 AUG 66. 06 2. 98 3. 88 79. 5 4. 7 0. 36 6. 44 1. 00 AGASSIZ SOILPM SOILZN SOILCU SOILPB SOILpH LEAFP LEAFZN LEAFCU DEC 50. 29 0. 60 1. 28 86. 3 5. 1 2. 38 10. 11 2. 35 DEC 37. 40 1. 37 6. 09 75. 0 5. 3 2. 51 11. 58 2. 14 DEC 48. 11 1. 08 1. 09 102. 5 5. 2 2. 54 11. 26 1. 98 DEC 26. 91 0. 57 0. 49 72. 5 5. 0 1. 52 8. 47 1. 00 FEB 30. 95 0. 81 1. 64 86. 9 4. 9 2. 52 16. 12 2. 79 FEB 36. 86 0. 52 1. 10 87. 6 5. 0 2. 43 16. 18 2. 52 FEB 41. 32 0. 86 0. 85 81. 9 4. 5 2. 69 20. 45 3. 42 FEB 17. 92 0. 27 0. 46 58. 2 4. 5 2. 42 20. 66 3. 57 MAY 41. 13 0. 60 1. 36 99. 4 4. 9 1. 91 6. 98 1. 00 MAY 49. 16 1. 14 2. 02 97. 5 4. 5 1. 76 7. 38 1. 00 MAY 55. 06 0. 85 1. 10 118. 8 5. 0 1. 59 7. 11 1. 28 MAY 39. 76 0. 60 0. 79 91. 9 4. 5 1. 38 6. 22 1. 54 JUN 26. 71 0. 74 2. 20 84. 4 4. 8 1. 50 7. 73 1. 65 JUN 28. 31 1. 07 3. 34 78. 2 4. 7 1. 49 8. 22 1. 44 JUN 37. 87 0. 93 2. 90 87. 6 4. 8 1. 56 7. 89 1. 54 JUN 24. 59 1. 05 3. 60 69. 4 4. 6 1. 62 8. 33 1. 80 AUG 32. 24 0. 91 2. 09 75. 5 4. 7 1. 29 8. 59 1. 00 AUG 34. 09 1. 72 2. 04 96. 5 4. 5 1. 26 8. 44 1. 00 AUG 117. 30 1. 97 2. 89 101. 5 4. 6 1. 14 8. 79 1. 00 AUG 38. 13 1. 08 1. 34 106. 0 4. 5 1. 07 7. 65 1. 00 91 APPENDIX I RESULTS OF PATTERNS STUDY A) S o i l and F o l i a r Data, con't CHILLIWACK SOILPM SOIL2N SOILCU SOILPB SOILpH LEAFP LEAFZN LEAFCU DEC 139. 2 1. 87 2. 95 150. 0 4. 7 2. 72 11. 25 2. 8 0 DEC 140. 6 0. 63 2. 73 145. 0 4. 9 2. 92 12. 0 3 3. 0 1 DEC 193. 1 0. 86 3. 25 190. 0 4. 7 2. 73 10. 59 2. 9 8 DEC 127. 7 1. 08 3. 36 140. 0 4. 7 2. 29 10. 53 2. 40 FEB 145. 7 0. 78 2. 25 161. 3 4. 6 2. 44 12. 40 3. 50 FEB 161. 8 0. 68 2. 53 156. 3 4. 3 2. 43 12. 11 3. 24 FEB 203. 4 0. 81 2. 84 176. 3 4.6 2. 65 12. 21 4. 19 FEB 204. 7 1. 15 2. 78 182. 5 4. 7 2. 47 12. 74 3. 29 MAY 28. 3 1. 99 3. 03 107. 5 4. 9 1. 23 6. 28 1. 30 MAY 149. 0 1. 07 2. 10 150. 0 4. 2 1. 21 6. 78 1. 00 MAY 148. 9 0. 76 2. 46 193. 6 4. 2 1. 93 7. 59 1. 0 0 MAY 168. 1 0. 98 2. 18 175. 0 4. 4 1. 90 8. 06 1. 00 JUN 131. 6 3. 07 7. 14 125. 0 4. 2 1. 32 5. 89 1. 0 0 JUN 121. 0 2. 56 7. 87 145. 0 4. 4 1. 32 4. 55 1. 00 JUN 139. 6 2. 25 5. 72 168. 8 4. 6 1. 56 6. 63 1. 00 JUN 129. 2 1. 99 5. 91 153. 8 4. 3 1. 46 6. 43 1. 0 0 AUG 166. 2 1. 83 3. 72 93. 0 4. 0 0. 67 16. 47 1 AUG 89. 4 1. 80 6. 11 88. 0 4. 2 0. 64 14. 45 1 AUG 115. 4 1. 94 3. 72 93. 0 4. 1 0. 58 14. 32 1 AUG 144. 2 1. 96 3. 11 142. 0 4. 1 0. 67 10. 63 1 DELTA SOILPM SOILZN SOILCU SOILPB SOILpH LEAFP LEAFZN LEAFCU DEC 115. 0 1. 28 7. 04 110. 0 5. 7 2. 49 11. 99 3. 28 DEC 140. 3 0. 62 7. 58 112. 5 5. 6 2. 29 9. 54 3. 49 DEC 108. 6 0. 51 7. 92 91. 3 5. 8 2. 40 10. 42 2. 91 DEC 132. 7 0. 63 8. 97 97. 5 5. 7 2. 63 12. 03 3. 59 FEB 146. 7 1. 94 6. 87 125. 0 5. 1 _ _ _ FEB 154. 9 0. 89 6. 62 155. 0 5. 5 - - -FEB 167. 5 1. 10 6. 95 137. 5 5. 6 - - -FEB 153. 7 1. 75 6. 68 140. 0 4. 9 - - -MAY 150. 5 1. 52 7. 08 118. 8 4. 9 2. 18 9. 59 2. 17 MAY 189. 5 1. 59 6. 66 167. 5 4. 7 2. 08 7. 57 2. 17 MAY 144. 8 2. 03 6. 11 80. 0 5. 1 1. 75 6. 98 1. 70 MAY 164. 1 2. 48 7. 06 137. 5 4. 7 1. 94 8. 60 1. 86 JUN 119. 7 1. 48 8. 45 127. 5 4. 6 1. 53 7. 58 1. 54 JUN 119. 7 1. 98 10. 50 103. 8 5. 0 1. 56 a. 70 1. 80 JUN 154. 1 4. 10 8. 59 115. 0 4. 8 1. 59 7. 44 2. 17 JUN 123. 4 1. 84 8. 27 82. 5 4. 4 1. 45 7. 77 1. 91 AUG 122. 5 1. 88 7. 13 84. 0 4. 7 0. 81 11. 36 1 . 00 AUG 95. 5 1. 88 7. 36 50. 0 5. 2 0. 79 13. 09 1 . 00 AUG 123. 2 2. 45 7. 61 79. 0 5. 2 0. 77 11. 20 1 . 00 AUG 124. 4 2. 10 8. 86 99. 0 4. a 1. 05 16. 30 1 . 00 _ - / _ . .. - - -92 APPENDIX I RESULTS OF PATTERNS STUDY B) Colonization Data CHILLIWACK DEC FEB MAY JUNE AUG 0 0 0 4 1 0 0 0 3 0 0 0 0 4 1 0 0 0 4 2 0 0 0 6 0 0 0 0 3 0 0 0 0 5 0 0 0 0 2 0 0 0 0 £ 0 0 0 0 3 0 0 0 0 4 0 0 0 0 3 0 0 0 0 2 0 0 0 0 3 1 0 0 0 3 0 0 0 0 3 2 0 0 0 3 0 0 0 0 5 2 0 0 0 3 0 0 0 0 3 0 0 0 0 4 0 0 0 0 3 0 0 0 0 4 0 0 0 0 5 0 DELTA DEC FEB MAY JUNE AUG 0 0 0 29 12 0 0 0 20 10 0 0 0 23 11 0 0 0 23 9 0 0 0 21 6 0 0 1 27 11 0 0 0 17 8 0 0 0 16 3 0 0 a 14 5 0 0 0 12 8 0 0 0 15 6 0 0 0 12 1 0 0 1 13 5 0 0 0 13 3 0 0 0 19 9 0 0 0 11 7 0 0 0 6 5 0 0 0 10 6 0 0 0 21 9 0 0 0 20 8 0 0 1 19 6 0 0 0 17 10 0 0 0 19 12 0 0 0 IB 10 OYSTER RIVER DEC FEB MAY JUNE AUG 0 5 40 40 2 0 1 35 40 2 0 2 38 53 2 0 3 23 38 6 0 1 24 31 3 0 3 30 29 0 2 2 28 33 2 1 3 20 38 2 1 3 31 45 2 2 2 23 48 2 1 2 19 50 0 1 3 28 42 1 3 1 14 26 0 1 0 8 35 0 1 0 12 26 2 1 0 18 24 0 1 1 16 23 2 1 0 21 26 1 2 1 20 34 1 1 0 17 19 1 1 1 9 19 1 3 2 12 20 1 AGASSIZ DEC FEB MAY JUNE AUG 1 0 10 19 0 1 3 11 17 0 1 1 14 30 2 0 0 9 21 3 0 1 19 23 1 2 1 12 29 5 2 0 9 24 2 1 0 16 14 3 1 1 16 25 2 1 1 14 17 6 1 1 18 20 6 1 1 22 26 7 1 0 13 21 2 1 1 15 23 4 1 3 16 22 4 1 0 7 17 4 1 1 9 19 0 1 0 14 20 9 1 1 17 25 7 1 0 11 21 2 1 0 11 25 5 1 0 7 24 7 2 2 9 21 4 2 2 13 18 3 2 1 5 23 6 2 1 8 23 6 93 APPENDIX II COLLECTION NUMBERS FOR FUNGAL SPECIES SMBBC # Species S i t e t. Date 73 Glgmus sp. 73 OR, Aug. * 74 Glomus monosgorum Gerd. & Trappe OR, Aug. 76 Sc l e r o c x s t l s rubiformis Gerd. & Trappe OR, Aug. 77 Glomus halonatum Rose & Trappe De, Aug. 78 Sclerocjjstie rubiformis Gerd. & Trappe De, Aug. 79 Acaulgsggra sp. 79 Ch, Aug 80 Sclerocy.st.is rubiformis Gerd. & Trappe Ch, Aug. 81 Glomus halonatum Rose & Trappe De, Dec. 82 Glgmus sp. 87 Ag, Dec. 83 Sc l e r o c y s t i s rubiformis Gerd. & Trappe OR, Dec. 84 Glomus monosgorum Gerd. & Trappe OR, Dec. 86 Acaulgsggra sp. 86 Ag, Aug. 87 Glomus sp. 87 Ch, Aug. 88 Glomus sp. 87 Ch, Dec. 89 Scutellosgora sp. 89 Ag, Dec. 90 Glomus halonatum Rose & Trappe Ag, Dec. 91 Glgmus sp. 91 Ag, Dec. * OR = Oyster River; De = Delta; Ch = Chilliwack; Ag = Agassiz; Aug. = August; Dec. = December. 94 APPENDIX III DATA FROM THE FUNGICIDE STUDY A) F o l i a r Data T r e a t m e n t D a y B C o l T C o l T r e a t m e n t D a y B C o l T C o l W 0 e I B W/0 0 1 0 1 0 W 0 6 1 9 W/0 0 7 5 W 0 8 2 4 W/0 0 6 8 W e> 5 2 6 W/0 0 5 1 0 w 0 11 2 0 W/0 0 8 7 w 0 1 3 1 4 W/0 0 9 9 w 0 4 9 W/0 0 9 7 w 0 3 11 W/0 0 6 8 w 0 4 1 0 W/0 0 8 2 w 0 e 1 3 W/0 0 5 4 w 0 8 11 W/0 0 2 3 w 0 5 6 W/0 0 8 3 w 0 3 4 W/0 0 5 11 w 0 3 3 W/0 0 4 1 2 w 0 2 6 W/0 0 4 7 w 0 3 3 W/0 0 9 2 w 0 2 2 W/0 0 4 7 w 0 2 0 W/0 0 8 1 3 w 0 2 8 W/0 0 5 3 w 0 1 5 W/0 0 4 5 w 0 2 5 W/0 0 5 5 w 0 1 6 W/0 0 5 3 w 0 3 1 0 W/0 0 5 6 w 0 2 3 W/0 0 9 7 w 4 9 1 6 W/0 4 8 5 w 4 8 1 7 W/0 4 6 5 w 4 8 1 9 W/0 4 5 6 w 4 1 0 2 0 W/0 4 3 6 w 4 11 1 5 W/0 4 5 3 w 4 1 2 1 9 W/0 4 5 8 w 4 5 1 4 W/0 4 4 8 w 4 4 21 W/0 4 6 8 w 4 2 11 W/0 4 4 6 w 4 5 1 3 W/0 4 a 9 w 4 4 1 0 W/0 4 4 5 w 4 4 11 W/0 4 4 1 0 w 4 7 7 w/o 4 6 1 5 w 4 4 4 W/0 4 3 8 w 4 3 4 W/0 4 2 1 2 w 4 5 6 W/0 4 4 7 w 4 4 4 W/0 4 5 1 2 w 4 5 4 W/0 4 9 8 w 4 2 4 W/0 4 4 3 w 4 2 7 W/0 4 3 3 w 4 2 9 W/0 4 4 4 w 4 2 8 W/0 4 4 1 w 4 5 8 W/0 4 1 2 w 4 5 8 W/0 4 2 0 w 6 11 9 W/0 8 6 1 0 w e 8 11 W/0 8 5 9 w e 6 1 6 W/0 8 9 5 w a a 7 W/0 a 8 4 w e 1 5 7 W/0 8 9 5 w e 1 3 5 W/0 8 1 0 4 w e 5 4 W/0 8 3 5 w a 4 3 W/0 8 6 ' 8 w a 9 3 W/0 8 7 4 w a 3 5 W/0 8 6 3 w 8 3 4 W/0 a a 3 w e 3 4 W/0 8 i i 3 w e 3 9 W/0 8 5 11 w e 2 8 W/0 8 7 1 6 w e 2 7 W/0 a 7 6 w e 2 7 w/o 8 6 1 7 w a 4 8 W/0 8 3 1 2 w a 2 8 w/o 8 7 1 9 w a 3 2 0 w/o 8 5 9 w e 2 1 6 w/o a 3 13 w 8 2 1 6 w/o a 7 8 w a 4 1 8 w/o a 4 5 w e 4 2 0 w/o e 6 5 w e 2 1 0 w/o 8 1 0 4 95 APPENDIX III DATA FROM THE FUNGICIDE STUDY A> F o l i a r Data, con't Treatment Day BCol TCol Treatment Day BCol TCol W 1 2 1 1 3 W/0 1 2 6 11 W 1 2 2 1 3 W/0 1 2 1 3 11 W 1 2 2 1 4 W/0 1 2 9 1 0 W 1 2 1 1 2 W/0 1 2 5 4 w 1 2 1 1 9 W/0 1 2 a 3 w 1 2 3 I B W/0 1 2 1 0 4 w 1 2 2 11 W/0 1 2 3 5 w 1 2 2 1 3 W/0 1 2 l 5 w 1 2 0 5 W/0 1 2 3 5 w 1 2 1 8 W/0 1 2 3 7 w 1 2 3 9 W/0 1 2 7 5 w 1 2 3 7 W/0 1 2 5 a w 1 2 1 2 6 w/o 1 2 1 1 5 w 1 2 0 1 4 W/0 1 2 4 1 8 w 1 2 3 1 0 W/0 1 2 7 1 2 w 1 2 1 1 2 W/0 1 2 4 1 5 w 1 2 0 1 2 W/0 1 2 4 1 6 w 1 2 1 1 2 W/0 1 2 8 11 w 1 2 0 2 W/0 1 2 3 9 w 1 2 0 4 W/0 1 2 1 1 3 w 1 2 0 4 W/0 1 2 6 6 w 1 2 0 3 W/0 1 2 4 3 w 1 2 1 1 W/0 1 2 6 6 w 1 2 0 5 w/o 1 2 8 7 w 1 6 e 1 5 W/0 1 6 4 1 2 w 1 6 e 1 0 w/o 1 6 7 9 w 1 6 6 1 2 w/o 1 6 7 1 0 w 1 6 1 2 a w/o 1 6 a 7 w 1 6 7 e w/o 1 6 a e w 1 6 1 0 9 w/o 1 6 5 1 0 w 1 6 1 0 1 8 w/o 1 6 2 1 2 w 1 6 3 1 3 w/o 1 6 3 1 7 w 1 6 1 1 5 w/o 1 6 5 1 2 w 1 6 5 1 1 W/0 1 6 4 1 4 w 1 6 6 1 2 w/o 1 6 6 11 w 1 6 7 1 2 w/o 1 6 5 14 w 1 6 1 9 w/o 1 6 6 14 w 1 6 1 1 5 w/o 1 6 4 17 w 1 6 2 1 0 w/o 1 6 9 21 w 1 6 2 1 0 w/o 1 6 a 2 2 w 1 6 2 1 0 w/o 1 6 a 1 5 w 1 6 S 9 w/o 1 6 a 1 6 w 1 6 1 4 w/o 1 6 5 2 w 1 6 1 1 w/o 1 6 7 1 w 1 6 1 2 w/o 1 6 4 2 w 1 6 2 1 w/o 1 6 5 4 w 1 6 3 3 w/o 1 6 8 3 w 1 6 3 3 w/o 1 6 1 0 3 w 2 0 a 8 w/o 2 0 1 0 6 w 2 0 1 2 7 w/o 2 0 1 0 5 w 2 0 5 1 0 w/o 2 0 5 0 w 2 0 7 4 w/o 2 0 7 5 w 2 0 s 9 w/o 2 0 a 6 w 2 0 3 5 w/o 2 0 1 3 4 w 2 0 7 11 w/o 2 0 4 5 w 2 0 4 1 0 w/o 2 0 6 1 0 w 2 0 e 1 0 w/o 2 0 3 3 w 2 0 e 1 2 w/o 2 0 5 9 w 2 0 5 9 w/o 2 0 3 6 w 2 0 6 1 2 w/o 2 0 2 6 w 2 0 5 3 w/o 2 0 6 5 w 2 0 3 8 w/o 2 0 7 3 w 2 0 5 9 w/o 2 0 5 7 w 2 0 5 6 w/o • 2 0 4 5 w 2 0 5 8 w/o 2 0 5 7 w 2 0 6 11 w/o 2 0 7 8 w 2 0 4 3 w/o 2 0 6 2 w 2 0 0 5 w/o 2 0 4 5 w 2 0 1 4 w/o 2 0 3 6 w 2 0 3 2 w/o 2 0 6 4 w 2 0 2 6 w/o 2 0 2 2 w 2 0 2 4 w/o 2 0 4 2 96 APPENDIX III DATA FROM THE FUNGICIDE STUDY B) Colonization Data Treatment Day W 0 W 0 W 0 W 0 W 4 W 4 W 4 W 4 w e w s w e w e W 12 W 12 W 12 W 12 W 16 W 16 W 16 W 16 W 20 W 20 W 20 W 20 B E o l P f BFolZn 3. 99 32. 18 4.23 28.47 4. 57 28. 38 3.99 21.49 2.97 22.21 3.47 22.17 3.48 21.80 3.46 17.75 2.65 15.99 2.66 15.58 2.85 16.08 2.58 11.92 2.05 11.53 2.39 13.79 2.66 15.52 2.43 12.71 2.25 14.70 2. 36 13. 44 2. 47 14. 53 2.17 11.95 2.07 13.43 2.25 13.78 2.65 15.52 2. 18 10. 52 l E g i P IEQIZQ 2.65 25.76 2. 90 46. 32 2. 63 55. 27 2.89 101.51 2. 52 20. 63 2.33 17.01 2. 30 18. 43 2. 59 23. 29 2. 42 30. 53 2. 22 28. 30 2. 22 26. 96 2. 10 22. 90 2. 09 19. 60 2.33 19.72 2.40 18.77 2.47 18.31 2.48 22.20 2. 54 20. 98 2.45 21.16 2. 51 20. 16 2. 22 20. 66 1.96 18.07 2.54 20.77 2. 50 20. 12 W/0 0 W/0 0 W/0 0 W/0 0 W/0 4 W/0 4 W/0 4 W/0 4 W/O 8 W/0 8 W/0 8 W/O 8 W/0 12 W/0 12 W/O 12 W/O 12 3.26 18.76 3.21 18.09 3. 22 18. 23 2.77 14.15 2.71 14.65 2.81 16.10 2.62 14.60 2.33 11.42 2.39 11.63 2. 16 10. 39 2.12 10.77 1. B1 10.94 2. 10 11. 04 2.20 12.05 2.03 11.50 1.93 9.36 3.01 24.53 2.81 23.46 2. 83 23. 89 2.43 19.96 2.34 18.34 2.46 17.13 2. 30 16. 84 2.57 22.55 2. 31 20. 94 2. 17 19. 51 2.01 18.71 2. 13 17.17 2.32 17.63 2.27 16.23 2. 15 15. 27 W/O 16 W/O 16 W/0 16 W/O 16 W/O 20 W/0 20 W/0 20 W/0 20 2.05 10.62 2.00 12.62 1.99 10.15 1.50 7.55 1.99 9.70 1.82 9.91 1.95 11.02 1.75 8.40 2. 52 20 22 2.24 18.24 2. 06 16. 00 2.23 17.83 2.44 20.65 2. 34 18. 55 2.40 19.79 2.24 17.34 • B = Bayleton t r i a l ; T = T i l t t r i a l ; FolP e foliar phosphorus; FolZn = foliar zinc; W • with fungicide; W/0 = without fungicide. 97 

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