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The effect of foliar applications of sprays made from kelp (macrocystis integrifolia) on growth of phaseolus… Radley, Reed Alan 1989

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THE EFFECT OF FOLIAR APPLICATIONS OF SPRAYS MADE FROM KELP {Macrocystis i n t e g r i f o l i a ) ON GROWTH OF Phaseolus v u l g a r i s : THE POSSIBLE ROLE OF ALGAL PHYTOHORMONE-LIKE SUBSTANCES by Reed Alan Radley B.Sc, The University of B r i t i s h Columbia A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Plant Science University of B r i t i s h Columbia We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 1989 © Reed Alan Radley 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 Plant Science The University of British Columbia Vancouver, Canada Date December 28, 1989 DE-6 (2/88) ABSTRACT The e f f e c t s of two applications of f o l i a r sprays made from kelp (Macrocystis i n t e a r i f o l i a Bory) on growth of beans (Phaseolus vulgaris L.) under three moisture regimes were investigated. Date of kelp harvest had l i t t l e e f f e c t on plants grown in water excess or d e f i c i t conditions, although means for kelp c o l l e c t e d i n spring tended to be higher. On s o i l s maintained near f i e l d capacity, spray made from kelp c o l l e c t e d i n July resulted i n increases i n some bean y i e l d components beyond the e f f e c t s of kelp c o l l e c t e d i n spring or f a l l . The e f f e c t of storage of l i q u e f i e d kelp at room temperature was also investigated and found to be s o i l moisture dependent. Leaf and root weights of plants grown i n f i e l d capacity and dry s o i l respectively were lower for plants treated with kelp aged for 118 days compared to plants treated with kelp stored for longer or shorter durations. In wet s o i l conditions, the e f f e c t of sprays on plant height was reduced by storage of the l i q u e f i e d kelp. Kelp extracts were fractionated by solvent p a r t i t i o n . The butanolic f r a c t i o n was shown to increase s i g n i f i c a n t l y many y i e l d variables under wet s o i l conditions. This f r a c t i o n had c y t o k i n i n - l i k e a c t i v i t y i n two bioassay systems, and contained substances co-eluting with isopentenyl adenine and zeatin i n column, thin layer, and g a s - l i q u i d chromatography systems. Non-s i g n i f i c a n t increases for some plant y i e l d components re s u l t i n g from application of unfractionated kelp p a r a l l e l e d these increases. A kelp f r a c t i o n with undefined constituents increased some y i e l d variables of plants grown i n f i e l d capacity s o i l , as did a f r a c t i o n with auxin and g i b b e r e l l i n -l i k e a c t i v i t i e s in bioassay. The presence of i n h i b i t o r s i n the kelp was indicated by s i g n i f i c a n t l y lower pod yiel d s under dry s o i l conditions, and a reduction i n cy t o k i n i n - l i k e bioassay a c t i v i t y when increasing amounts of kelp were assayed. The p o s s i b i l i t y that phytohormone d e f i c i t s r e s u l t i n g from root stress are ameliorated by some components of the kelp spray i s discussed. i i i TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i LIST OF ABBREVIATIONS ix ACKNOWLEDGEMENTS x 1 INTRODUCTION 1 1.1 SEAWEEDS AS MANURES 2 1.2 SEAWEED FOLIAR SPRAYS 6 1.3 SEED TREATMENT WITH SEAWEED EXTRACTS 9 1.4 EFFECTS OF FOLIAR APPLICATIONS 12 1.4.1 Y i e l d 12 1.4.2 Tissue Composition 20 1.4.3 Development and Dry Matter P a r t i t i o n i n g 26 1.4.4 Storage Quality 32 1.4.5 Disease and Pest Resistance 35 1.5 MECHANISM OF ACTION OF SEAWEED FOLIAR SPRAYS 38 1.5.1 N u t r i t i o n a l Hypothesis 38 1.5.2 Phytohormonal Hypothesis 42 2 MATERIALS AND METHODS 52 2.1 KELP COLLECTION 52 iv 2.2 KELP PROCESSING 53 2.3 GREENHOUSE EXPERIMENTS 54 2.3.1 Date of Kelp Harvest 55 2.3.2 Shelf L i f e 57 2.3.3 Fractionation of Kelp Extract 58 2.4 PHYTOHORMONE ANALYSIS 61 2.4.1 Bioassays 62 2.4.2 Phytohormone Extraction and P u r i f i c a t i o n 67 2.4.3 Phytohormone Chromatography 7 6 3 RESULTS 82 3.1 GREENHOUSE EXPERIMENTS 82 3.1.1 Date of Kelp Harvest 82 3.1.2 Shelf L i f e 84 3.1.3 Kelp Fractionation 88 3.2 PHYTOHORMONE ANALYSIS 103 3.2.1 Analysis of Monthly Samples 103 3.2.2 Additional Analysis of Seaweed Collected i n May 129 4 DISCUSSION 138 5 SUMMARY/CONCLUSIONS 153 6 BIBLIOGRAPHY 157 v LIST OF TABLES Page Table 1 Commercial f o l i a r seaweed sprays 8 Table 2 Elemental content of beet (Beta vulgaris) crop compared to elemental content of f o l i a r l y applied seaweed preparations 39 Table 3 Effe c t of kelp spray made from Macrocystis integrifolia c o l l e c t e d at various times of the year on growth of Phasiolus vulgaris under three moisture regimes 83 Table 4 Ef f e c t of kelp spray made from Macrocystis integrifolia stored at 20°C on the growth of Phasiolus vulgaris under three moisture regimes 85 Table 5 Ef f e c t of f o l i a r sprays made from extracts of M. integrifolia on bean growth under three s o i l moisture regimes 90 Table 6 Auxin-like a c t i v i t y of extracts of Macrocystis integrifolia 110 Table 7 Cytokinin-like a c t i v i t y of extracts of Macrocystis integrifolia 119 Table 8 Co-elution and bioassay responses of extracts of Macroystis integrifolia 136 vi LIST OF FIGURES Page Figure 1 Flow diagram of the extraction methods of Rademacher and Graebe (1984) 70 Figure 2 Flow diagram of the extraction methods of Mukherji and Waring (1983) 73 Figure 3 Flow diagram of the extraction methods of Taylor et al. (1982) 75 Figure 4 Recovery of IAA following extraction and p u r i f i c a t i o n 104 Figure 5 Recovery of IAA following t h i n layer chromatography 10 6 Figure 6 Recovery of isopentenyl adenine (IPA) and isopentenyl adenosine (IPAR) following extraction and p u r i f i c a t i o n 107 Figure 7 Recovery of isopentenyl adenine (IPA) and isopentenyl adenosine (IPAR) following chromatography on Dowex 50X 108 Figure 8 Auxin bioassay of extract of 100 g May seaweed I l l Figure 9 Auxin bioassay of extract of 100 g May seaweed 112 Figure 10 Auxin bioassay of extract of 100 g July seaweed 113 Figure 11 Auxin bioassay of extract of 100 g July seaweed 114 Figure 12 Auxin bioassay of extract of 100 g September seaweed 115 Figure 13 Auxin bioassay of extract of 100 g September seaweed 116 Figure 14 Cytokinin bioassay of extract of 100 g May seaweed 121 v i i Figure 15 Cytokinin bioassay of extract of 100 g May seaweed 122 Figure 16 Cytokinin bioassay of extract of 100 g July seaweed 123 Figure 17 Cytokinin bioassay of extract of 100 g July seaweed 124 Figure 18 Cytokinin bioassay of extract of 100 g September seaweed 125 Figure 19 Cytokinin bioassay of extract of 100 g September seaweed 12 6 Figure 20 G i b b e r e l l i n bioassay of extract of 100 g May seaweed 128 Figure 21 Cytokinin bioassay of extract of 5 g May seaweed 130 Figure 22 Cytokinin bioassay of t r i f l u o r o a c e t i c acid-hydrolized extract of 10 g May seaweed 132 Figure 23 Cytokinin bioassay of 1, 10, and 40 g May seaweed 134 Figure 24 Results of co-chromatography of XAD 2 eluates with authentic cytokinins 137 v i i i LIST OF ABBREVIATIONS ABA absc i s i c acid BA benzylaminopurine BSTFA b i s - t r i m e t h y l s i l y l trifluoracetamide CCC chlormequat chloride D dry (-120 to -150 kPa) EtOAc ethyl acetate EtOH ethanol FC f i e l d capacity (-30 to -50 kPa) FID flame i o n i s a t i o n detector GA g i b b e r e l l i n GA3 g i b b e r e l l i c acid GLC gas - l i q u i d chromatography HPLC high performance l i q u i d chromatography IAA indoleacetic acid IPA isopentenyl adenine IPAR isopentenyl adenosine K kin e t i n LA leaf area LAR leaf area r a t i o LSD least s i g n i f i c a n t difference MeOH methanol NAA naphthylacetic acid PVP polyvinylpyrrolidone RT retention time RUBPC ribulose biphosphate carboxylase SLA s p e c i f i c leaf area TFA t r i f l u o r o a c e t i c acid TLC t h i n layer chromatography W wet (0 to -10 kPa) Z zeatin ZR zeatin riboside ix_-ACKNOWLEDGEMENT S I would l i k e to g r a t e f u l l y acknowledge the moral support of my parents and friends (Bunny) throughout t h i s exercise. Thanks are also due Brian Holl for his support and patience, to Wayne Temple for many discussions, and to I.E.P. Taylor for kindly providing lab space i n the i n i t i a l stages. Many thanks also to my committee members for speedily dealing with my defence. The f i n a n c i a l support of the Science Council of B.C. i s also acknowledged. 1. INTRODUCTION SEAWEEDS IN AGRICULTURE Seaweeds have been used to enhance crop production i n v i r t u a l l y a l l coastal areas of the world for centuries. According to Booth (1953, 1963), the ancient Greeks, Romans, Chinese and Vikings a l l applied seaweed to the s o i l to increase yields of cul t i v a t e d plants. In France, seaweed was considered such an important f e r t i l i z e r resource that the king issued a decree i n 1681 regulating i t s harvest and use. Similarly, along the I r i s h coast, seaweeds were considered more valuable than conventional manures. In fact, farmers . marked out sections of wild kelp beds with rocks to delineate t h e i r areas and reserve t h e i r kelp supply. These common law rights of harvest persisted into the 19th century (Bell and Watson, 1986). In the Aran Islands i n Galway, p i l i n g alternate layers of seaweed and sand was termed "making land". More recently, the use of kelp as a f e r t i l i z e r resource has been stimulated to large scale by the threat of war; in 1917-18, 10% of the potash requirement of the United States was supplied by kelp from C a l i f o r n i a . The e a r l i e s t formal report of experiments involving the use of seaweed f e r t i l i z e r s was published i n 1942 i n response to the wartime threat to New Zealand's imported potash supply. 1 In addition to the use of seaweeds as s o i l amendments, l i q u i f i e d seaweeds, seaweed extracts and seaweed hydrolysates have been used as f o l i a r sprays and seed treatments to t r y to enhance yields on a number of crops. Two main hypotheses have been proposed to account for the effects of these preparations on plants. One i s that the response i s due e n t i r e l y to the n u t r i t i o n a l content of the seaweed spray: however, the application rate of such preparations i s generally so low that l i t t l e contribution i s l i k e l y to be made to the n u t r i t i o n a l status of the crop. The other main hypothesis i s that the seaweed preparations contain phytohormonal substances that stimulate increased growth of the crop. This contention i s supported by the e f f i c a c y of very low application rates, and the nature of some of the reported crop responses. The objectives of the work described here were to determine whether a coastal B r i t i s h Columbia seaweed, Macrocystis i n t e g r i f o l i a had the potential for development into an e f f e c t i v e f o l i a r spray as the basis of a viable l o c a l industry, to document the eff e c t of such a f o l i a r spray on Phaseolus vulgaris, and to evaluate the source of the b i o l o g i c a l a c t i v i t y of the f o l i a r spray. 2 1.1 SEAWEEDS AS MANURES Despite the long history of the seaweed use i n agriculture, reports i n the l i t e r a t u r e are few. An early report by the Dominion of Canada, Department of Agriculture reported elemental analysis of kelp and suggested that seaweed would be a good source of potassium for a g r i c u l t u r a l production (Shutte and Wright, 1927). The report c a l l e d seaweeds "complete f e r t i l i z e r s " comparing favourably with t r a d i t i o n a l manures, and pointed out several added advantages including no weed seeds, fungal or pest associations, and no requirement for composting. Rapson et a l . (1942) car r i e d out f i e l d t r i a l s i n New Zealand that compared seaweed and elementally equivalent amounts of potash f e r t i l i z e r . Although no numerical data were reported, the authors concluded that the y i e l d responses to seaweed at rates equivalent to 30% potash s a l t s were only s l i g h t l y i n f e r i o r to those i n chemically f e r t i l i z e d p l o t s . They concluded that, should the need arise, seaweed could supply a portion of New Zealand's f e r t i l i z e r needs, and recommended seaweed use i n coastal regions. Francki (1960) applied seaweed meal at a rate of 1.25% (w/w) to potted s o i l i n which he grew tomatoes (Lycopersicon  esculentum). He found that the red alga, Pachvmenia spp., could be detrimental or stimulatory to plant growth, 3 depending on the s o i l type. Durvillea, a brown alga, was i n h i b i t o r y on a l l s o i l types tested. Plants suffered i n i t i a l i n h i b i t i o n with subsequent p a r t i a l recovery. Francki (1960) also found that pre-incubating the seaweed/soil mixtures reduced the detrimental e f f e c t s , which he attributed to an i n i t i a l nitrogen deficiency r e s u l t i n g from a high C/N r a t i o (Pachymenia, 20; D u r v i l l i a , 35), or to s a l t t o x i c i t y . In other experiments i n which plants with a wide range of s a l t tolerances were grown in seaweed-treated s o i l , Francki reported that s a l t tolerant species were most adversely affected. Furthermore, s a l t i n amounts equivalent to those introduced to the s o i l i n seaweed f a i l e d to depress plant growth. These results indicated that s a l t t o x i c i t y was not the primary cause of y i e l d losses. Francki (1960, 1964) also noted that seaweed had s i g n i f i c a n t e f f ects on s o i l physical properties that ultimately could affect mineral a v a i l a b i l i t y , and concluded that "the manurial values of seaweeds ... should not be based purely on the chemical analysis of a species". Although the results described above are not p a r t i c u l a r l y encouraging, other papers present data showing generally favorable responses to seaweed f e r t i l i z e r application. Milton (1964) reported an i n i t i a l depression or "latent period" immediately following seaweed application, s i m i l a r to that described by Francki (1960), but subsequently followed by 4 manifestation of f e r t i l i z e r e f f e c t s . He attributed the negative effects of D u r v e l l i a meal application i n part to microbial p r o l i f e r a t i o n and b i o l o g i c a l immobilization of nitrogen as a consequence of i t ' s high C/N r a t i o . Pachymenia. a seaweed with a narrower C/N r a t i o , did not e l i c i t N-. . . 2 + deficiency symptoms in pot experiments; however Mg concentration in crop foliage was at near toxic l e v e l s on some 2 + s o i l s . Francki (1964) also noted high levels of Mn i n seaweed-treated s o i l s caused by the release of available s o i l 2+ . Mn i n response to waterlogging. More recently, Temple and Bomke (1988) conducted experiments to determine the e f f e c t of various application rates of the brown seaweed Macrocystis i n t e a r i f o l i a on bean (Phaseolus vulgaris) y i e l d and nutrient status, and s o i l chemical and physical properties. They found that application of fresh seaweed at rates up to 60 t ha had a p o s i t i v e e f f e c t on bean y i e l d s . Negative effects at higher rates were attributed to s a l t t o x i c i t y , based on symptoms and tissue analyses. They concluded that the b e n e f i c i a l effects of seaweed manure were exerted not only through the addition of mineral nutrients to the s o i l , but through improved s o i l structure. Alginates, agars and fucoidins constitute up to 30% of kelp dry weight. These al g a l carbohydrates which are used extensively as binders and s t a b i l i z e r s i n the food industry, may also contribute to improved s o i l structure. 5 Furthermore, seaweeds also have the a b i l i t y to chelate certain micronutrients which may also contribute to s o i l improvement. Oppermans C a s t i l l o (1966, c i t e d i n Booth, 1969) compared the effects of seaweed f e r t i l i z e r with equivalent a l g i n i c acid treatments on plant growth i n a calcareous s o i l . A l g i n i c acid increased the iron a v a i l a b i l i t y i n the s o i l , but not as much as the seaweed treatments. More comprehensive experiments by Blunden and Woods (1969) tested the effects of the main carbohydrate constituents of seaweed on growth of mustard (Sinapis alba) in Vermiculite. They found that mannitol increased the wet weights of test plants, but laminarin and carageenin reduced them. In addition, 0.05% (w/w) potassium alginate in s o i l increased the mean fresh weights of test plants, but not as much as the equivalent amount of inorganic potassium s a l t . These experiments, however, were designed to test for dire c t b i o l o g i c a l a c t i v i t y , rather than i n d i r e c t e f f e cts mediated through improvements i n s o i l structure, and demonstrated that no single a l g a l carbohydrate can acount for the growth improvement r e s u l t i n g from seaweed application. Thus, i n cases where seaweed has proved b e n e f i c i a l when applied as a f e r t i l i z e r , the benefits seem to arise from two main a l g a l properties: 1) Addition of mineral elements to the s o i l (ash content 15-25%, compared to 5% i n hay (Kingman and Senn, 1978) ) ; 2) Addition of organic matter to the s o i l which improves s o i l structure and mineral n u t r i t i o n through 6 micronutrient chelation and increased microbial a c t i v i t y . 1.2 SEAWEED FOLIAR SPRAYS It i s well established that foliage, as well as roots can provide a path of entry and egress for plant nutrients (Wittwer et a l . , 1969) The f i r s t report of curing iron deficiency through f o l i a r application dates from 1844 (Gris, c i t e d in Wort, 1967) . The use of radio-isotopes allowed v e r i f i c a t i o n of the entry of mineral nutrients through leaf surfaces. Stephenson (1968) attributed the f i r s t f o l i a r applications of seaweed to Dr. R. Milton, who was working to develop seaweed-based t e x t i l e s during World War II. An avid gardener, he also t r i e d various ways of tre a t i n g seaweed to l i q u i f y seaweed and thus make i t "more e a s i l y assimilated" by his garden crop. Since that time several seaweed-based products have been manufactured and marketed (Table 1). When f o l i a r application of nutrients and growth regulators became an accepted a g r i c u l t u r a l practice during the 1960's, i t provided a r a t i o n a l basis for the growth promoting properties claimed by seaweed spray manufacturers. This acceptance of f o l i a r f e r t i l i z a t i o n in turn prompted an increased demand for these products based on extraordinary manufacturers' claims 7 TRADE NAME SEAWEED USED ORIGIN Nitrozyme/Algit/ Algifert/Algospray/ Marinure Ascophyllum Norway Chase SM-3/ Cytex Ascophyllum Fucus/Laminaria B r i t a i n Maxicrop Ascophyllum B r i t a i n Sea-sol Durvillia A u s t r a l i a Goe-Mar Laminaria France Kelpak Ecklonia South A f r i c a TABLE 1 Commercial F o l i a r Seaweed Sprays Data are from Temple et al., 1986. 8 that seaweed products could: i) increase y i e l d and quality of f i e l d and h o r t i c u l t u r a l crops i i ) increase shelf l i f e of various f r u i t s i i i ) combat pests and disease iv) increase resistance to frost and other environmental stresses v) improve mineral n u t r i t i o n without added f e r t i l i z e r vi) increase seed germination and r e s p i r a t i o n v i i ) stimulate s o i l microbial a c t i v i t y v i i i ) increase the rooting of cuttings, and ix) prolong the quality of cut flowers. In short, according to manufacturers' claims, there was l i t t l e that seaweed sprays could not do. The s c i e n t i f i c community responded to these wide-ranging claims with skepticism, since they were often based either on poorly controlled experiments performed by the manufacturers themselves, or on anecdotal testimonials from s a t i s f i e d customers. Despite t h i s apparent s c i e n t i f i c r e j e c t i o n of seaweed f o l i a r sprays, occasional reports i n the l i t e r a t u r e have appeared which indicate that seaweed sprays can indeed aff e c t the growth and n u t r i t i o n of plants. 9 1.3 SEED TREATMENT WITH SEAWEED EXTRACTS Several authors have reported increases i n germination rate of seeds soaked i n seaweed preparations. Early experiments by Button and Noyes (1964) indicated that 0.5% and 1% solutions of SM-3, a B r i t i s h seaweed product used to soak creeping red fescue seeds (Festuca spp.), increased the number of emerged seedlings by 63% af t e r 1 week and 7 9% after 2 weeks. Higher concentrations were inhi b i t o r y , and no germination occurred at 18% seaweed extract i n water. Aitken and Senn (1965) and Senn and Skelton (1969) reported that 1:25 to 1:50 d i l u t i o n s of seaweed preparation i n water stimulated germination of zinnia (Zinnia elegans), tobacco (Nicotiana tabacum), peas(Pisum  sativum), radish (Raphanus sativus), cotton (Gossypium  herbaceum), white (Pinus alba) and l o b l o l l y pine (Pinus  taeda), h o l l y (Ilex opaca), ligustrum (Ligustrum lucidum) and nandia (Nandia domesticum). They noted an inverse relationship between seed re s p i r a t i o n rate and concentration of seaweed extract although no data, experimental design, or significance l e v e l s were given. Wilczek and Ng (1982) wrote that a 12-hour soak i n 1% Cytex, a commercial seaweed extract, increased beet (Beta  vulgaris) germination rates by up to 50% compared to water-treated controls. They also reported an interaction between 10 temperature and seaweed extract concentration on promotion of germination at 10°, 15°, 20°, and 30°, but i n h i b i t i n g germination by 21% at 25°. Data on ultimate per cent germination were not presented. In experiments with Pinus  radiata, Donald (1981) found that soaking seeds i n a 1:500 d i l u t i o n of Kelpak i n water for 24 hours p r i o r to s t r a t i f i c a t i o n decreased dormancy s i g n i f i c a n t l y . Goh (1971) applied kelp extract to white clover (Trifolium repens) seeds on f i l t e r paper and i n three d i f f e r e n t s o i l types. He found that germination rate and ultimate per cent germination were increased by up to 17% on one low f e r t i l i t y s o i l ; however t h i s peak at eight days afte r sowing had declined to 4% by day 12. The seaweed treatments had no ef f e c t on germination on f i l t e r paper, and reduced germination on s o i l contaminated with persistent a g r i c u l t u r a l chemicals (e.g. re s p i r a t i o n i n h i b i t o r s ) . F i n a l l y , Stephenson (1973) reported 2.5% and 4% increases i n germination rates of winter wheat (Triticum  aestivum) and barley (Hordeum vulgare) seed respectively, for seeds taken from seaweed treated plants. Since germination rate can be a s i g n i f i c a n t factor i n securing establishment (Derwyn et a_l., 1966), the d i r e c t e f f e cts of seaweed on seed germination are of i n t e r e s t . In addition to these studies concerned with germination, others have been conducted to determine the effects of seed treatment on subsequent growth of the plants. Miers and Perry (1986) treated wheat seeds with a seaweed product, Seasol, and reported no evidence of any p o s i t i v e e f f e c t on y i e l d i n extensive f i e l d t r i a l s ; germination rates were not recorded. Featonby-Smith and van Staden (1987) recently treated barley seed with Kelpak (1 hour, 1:500 dilution) and reported increased grain mass of the r e s u l t i n g potted plants, but no eff e c t on in d i v i d u a l kernel mass. Y i e l d increases were the result of an increased number of ears and increased percentage of f e r t i l e spikelets on plants growing from seaweed-treated seeds. Germination and emergence data were not reported. 1.4 EFFECTS OF FOLIAR APPLICATION 1.4.1 Yi e l d : Reports of experiments to test crop responses to f o l i a r application of seaweed sprays are numerous, as are unreplicated anecdotal reports from private c i t i z e n s , farmers and h o r t i c u l t u r a l i s t s . Many of these claimed substantial y i e l d increases as a result of seaweed application. ' Stephenson (1966) reported t r i a l s in Bedfordshire i n -1 which 1:120 d i l u t i o n s (2 gal acre ) of Maxicrop applied at three week interv a l s increased brussel sprout (Brassica  oleracea var. qemmifera) y i e l d by 12%, and by 32% when applied with the i n s e c t i c i d e Metasystox. S t a t i s t i c a l d e t a i l s were not given. He also described two t r i a l s performed on blackcurrents (Ribes nigrum) on government experimental stations. These experiments gave 12% and 27% y i e l d increases following treatment of bushes with 1:400 d i l u t i o n s (1.5 gal acre 1) at two week int e r v a l s beginning at the open flower stage. Again, no s t a t i s t i c a l analysis was reported. Aitken and Senn (1965), as part of an ongoing interest at Clemson University i n the use of seaweeds i n agriculture since 1958, reported an increase i n y i e l d of grapes ( V i t i s  v i n i f e r a ) after f o l i a r application of seaweed extract. The treatments, application rates and significance levels were not reported. Goh (1971) treated white clover plants with seaweed spray i n experiments on three d i f f e r e n t s o i l types. He reported no y i e l d e f f e c t of seaweed applied at 4, 12, or 20 gal. acre 1 on any s o i l . Experiments at Clemson (Senn and Kingman, 1978) using weekly applications of alkali-hydrolyzed extracts of Ascophyllum resulted in no eff e c t on soybean (Glycine max), okra (Hibiscus esculentus), tomato or southern pea, whereas growth of corn (Zea mays) growth was reduced 7%. Stephenson (1966) reported a 15-32% s t a t i s t i c a l l y s i g n i f i c a n t y i e l d increase of strawberries (Fragaria spp.)in three t r i a l s , and turnip (Brassica rapa) y i e l d increases of 23%. In a three year study to extend an e a r l i e r report (Povolny, 1966) in which a 41% y i e l d increase in cucumber (Cucumis sativus) y i e l d was described, Povolny (1968) sprayed 0.04% and 0.08% A l g i f e r t on gherkins. Plants were sprayed 6-10 times with 7-10 1 acre of seaweed concentrate. He obtained s t a t i s t i c a l l y s i g n i f i c a n t y i e l d increases of from 10 to 122%. In another experiment with cucumber, Nelson and van Staden (1984a) tested the ef f e c t of a weekly f o l i a r spray of Kelpak (1:500 d i l u t i o n , sprayed u n t i l runoff). They obtained s t a t i s t i c a l l y s i g n i f i c a n t increases in root (96%) and t o t a l dry matter (55%) i n "nutrient-stressed" plants. They noted an i n i t i a l i n h i b i t i o n of f r u i t growth, but did not report f i n a l f r u i t production. Further experiments by Povolny (1976) between 1972 and 1975 demonstrated that sprays of 0.5% and 1% of Algae Produkter seaweed product applied at 10-14 day in t e r v a l s , 4-8 times increased tomato y i e l d by 15-29%. Stephenson (1973) recorded increases of 8% i n winter -1 wheat to which f i v e pints acre of Maxicrop were applied, -1 and 11% when one gal. acre was applied. Although the treatments were replicated, no treatment comparisons with significance levels were given. In the same publication, increased i n t u r f production was reported following a single -1 treatment with f i v e pints acre of Maxicrop. Twenty-one days afte r spraying, biomass of sprayed plants was 15.7% greater than controls. After 42 days, sprayed t u r f had regrown 11.8% more than controls, but by 63 days afte r spraying, regrowth was similar i n sprayed and unsprayed p l o t s . He also reported that treatment of tomato seedlings at transplant and six times thereafter with a 1:200 d i l u t i o n of Maxicrop resulted i n a 37% y i e l d increase. Csizinszky -1 (1984) applied 2.5 1 ha of seaweed spray to tomato plants and observed a 20% increase i n t o t a l y i e l d and a 17% increase in marketable y i e l d after three sprays at 2 week i n t e r v a l s . Blunden (1972) reported that SM-3 increased the bunch weight of Jamaican bananas (Musa spp.). The largest increases, 22%, followed 2 applications of 0.75 gal. acre 1 . -1 Peppers (Capsicum annuum) sprayed at 0.5 and 1 gal. acre at the f i r s t blossom stage increased y i e l d by 6.3 and 26.6% respectively. 'La Soda' potatoes (Solanum tuberosum) receiving a 1% spray at blossom increased potato y i e l d by -1 37%, while corn (Zea mays) sprayed with 0.5 gal. acre twice at three week interv a l s yielded 56% more ripe ears i n addition to being t a l l e r , thicker and greener. F i n a l l y , he also reported a 10% increase i n orange (Citrus sinensis) -1 y i e l d r e s u l t i n g from a 1 gal. acre application. Unfortunately, Blunden (1972) did not provide the significance l e v e l s associated with the increased mean yields , and some of these t r i a l s were not s t a t i s t i c a l l y v a l i d . Blunden and Wildgoose (1977), i n well-designed experiments, tested the effects of SM-3 at two d i l u t i o n rates (100 and 200-fold) on two potato v a r i e t i e s , 'King Edward' and 'Pentland D e l l ' . Plants were sprayed once, 6 weeks after emergence. 'King Edward' potatoes yielded a s i g n i f i c a n t 1 2 . 9 % g r e a t e r t u b e r w e i g h t t h a n t h e c o n t r o l a t t h e h i g h e r a p p l i c a t i o n r a t e . A 1 2 . 2 % i n c r e a s e i n ' P e n t l a n d D e l l ' y i e l d was n o n - s i g n i f i c a n t . F u r t h e r t r i a l s t h e f o l l o w i n g y e a r showed an 1 8 . 5 % i n c r e a s e i n t u b e r y i e l d , w i t h t h e i n c r e a s e due m o s t l y t o an i n c r e a s e i n t h e p r o p o r t i o n o f ware ( l a r g e ) p o t a t o e s . More r e c e n t e x p e r i m e n t s w i t h p o t a t o e s w e re c o n d u c t e d b y D w e l l e a n d H u r l e y (1984) a n d L a n g a n d L a n g v i l l e ( 1 9 8 4 ) . The f o r m e r a u t h o r s c o n d u c t e d a s e v e n - y e a r s t u d y on t h e u s e o f Cytex (= SM-3) on s e v e r a l p o t a t o c l o n e s . They t e s t e d v a r i o u s a p p l i c a t i o n r a t e s a n d d a t e s , a n d o b s e r v e d no r e s p o n s e o f ' R u s s e t B u r b a n k ' t o e i t h e r Cytex o r k i n e t i n . ' L e h mi R u s s e t ' showed s i g n i f i c a n t r e s p o n s e s ( 2 5 % , 9%, 10%) i n t h r e e o f f i v e y e a r s . ' B u t t e ' p o t a t o e s showed a 3 5 % i n c r e a s e i n one o f t h r e e y e a r s . L a n g a n d L a n g v i l l e (1984) v e r i f i e d t h e l a c k o f a r e s p o n s e t o Cytex by ' R u s s e t B u r b a n k ' , h o w e v e r t o t a l y i e l d o f 'Kennebec' p o t a t o e s w e re i n c r e a s e d 16% i n r e s p o n s e t o a s i n g l e a p p l i c a t i o n o f 0.15% seaw e e d e x t r a c t . - i I n 197 9, B l u n d e n e t a l . . u s e d SM-3 a t 4.5 1 a c r e on v a r i e t i e s o f s u g a r b e e t ( B e t a v u l g a r i s ) . The seaw e e d was a p p l i e d o n c e , a n d r e s u l t e d i n a s i g n i f i c a n t 8.6% i n c r e a s e i n r o o t w e i g h t i n o n l y one o f t h r e e y e a r s . D e V i l l i e r s e t a l . (1983) c o m p a r e d t h e e f f e c t s o f two se a w e e d p r o d u c t s i n t r i a l s w i t h ' G o l d e n D e l i c i o u s ' a n d ' S t a r k i n g ' a p p l e ( P y r u s m a l u s ) t r e e s , 'Van R i e b e e k ' p e a c h ( P r u n u s p e r s i c a ) t r e e s , a n d ' A l p h o n s e L a v a l l e e ' a n d 'Barlinka' table grapes. The t r i a l s took place in South A f r i c a over two seasons, and 1:500 d i l u t i o n s were applied three times i n December and January the f i r s t year, and at three-week in t e r v a l s beginning at bud-break the second year. A non-significant incease (7.5%) i n mean apple yields was recorded. A decrease i n grape y i e l d of 8.2% was also non-s i g n i f i c a n t . There was no e f f e c t on peach y i e l d . Luanratana and G r i f f i n (1980a,b) showed that spraying Duboisia trees with 0.5% Maxicrop solution increased the leaf y i e l d by 18% i n the f i e l d , but had no e f f e c t on mass of hydroponically grown plants. Lettuce (Lactuca sativaV in the f i e l d receiving 3, 6, or -1 9 1 ha of Maxicrop every two weeks for 10 weeks showed 8.6% - i greater y i e l d over controls at the 6 1 ha rate. No e f f e c t of 0.3, 0.6, or 1.2% Maxicrop was reported when i t was applied to cauliflower (Brassica oleracea var. botrytis) (Abetz and Young, 1980). Kotze and Joubert (1980) reported no shoot weight increases as a result of spraying three leve l s of seaweed onto cabbage (Brassica oleracea) i n either high or low f e r t i l i z e r l e v e l s o i l . Root growth, however, was doubled by the 1:330 d i l u t i o n , increased 50% at 1:500 and 20% by the 1:1000 d i l u t i o n . Several investigators have looked at the effects of seaweed f o l i a r sprays on grain crops. As mentioned above, Stephenson (1973) reported s i g n i f i c a n t 8-11% y i e l d increases 17 from winter wheat sprayed with seaweed extract. Nelson and van Staden (1984) applied Kelpak to wheat i n a growth chamber. One hundred-fold d i l u t i o n s were applied weekly for eight weeks, and a number of p o t e n t i a l l y b e n e f i c i a l results were recorded, including y i e l d increases of 67%. A l a t e r study from the same laboratory tested the effects of Kelpak on barley i n a growth chamber. F o l i a r sprays of 20 ml of 1:250 or 1:500 were applied two weeks after emergence. Grain mass per plant was increased 54% by the 1:250 spray and 51% by the 1:500 spray (Featonby-Smith and van Staden, 1987). Kotze and Joubert (1980) reported a favourable ef f e c t of seaweed spray application to rye. W e l l - f e r t i l i z e d rye (Secale cereale) plants in pots responded to 1:500 and 1:1000 di l u t i o n s of l i q u e f i e d seaweed with 50% and 85% increases i n y i e l d respectively. At higher application rates (1:330), or in u n f e r t i l i z e d s o i l , f o l i a r spraying had no ef f e c t on shoot dry weight. Root growth i n f e r t i l e s o i l was doubled by spray d i l u t e d to 1:330 and 1:1000, but was unaffected by a 1:500 d i l u t i o n . In Canada, f i e l d experiments using Nitrozyme as a seed -1 treatment (0.3 1 ton ) and f o l i a r spray at the four leaf stage had no ef f e c t on y i e l d of 'Harrington' barley. 'Bonanza' barley was s i g n i f i c a n t l y i n h i b i t e d by Nitrozyme i n -1 plots that also received N at 40 kg ha , but had no e f f e c t -1 in plots f e r t i l i z e d with 80 kg ha . Si m i l a r l y no e f f e c t on 18 "robin' or 'Westar' canola (Brassica campestris) yields was -1 noted i n response to f o l i a r treatment (1 1 ha Nitrozyme) at the 3-4 leaf stage and at bol t i n g (Taylor and Foster, Agriculture Canada Research Station, Lacombe Alberta, personal communication,1986). More extensive t r i a l s involving 32 replicated f i e l d t r i a l s over a three year period were conducted i n Western A u s t r a l i a to evaluate various b a c t e r i a l , f i s h , and seaweed products. Two seaweed products, Seasol and Kelpak were -1 tested. In 1981, Kelpak at 0.2 1 ha caused s i g n i f i c a n t y i e l d depression (9%) at one of the s i t e s . During 1982 and 1983 Kelpak applied at various rates and times at two si t e s had no eff e c t on y i e l d , and no eff e c t on the mean y i e l d across 16 diverse t r i a l s i n 1982 or nine t r i a l s i n 1983. It i s noteworthy that none of the products tested, including such common and d i s s i m i l a r treatments as Complesal, an inorganic nutrient f o l i a r spray, Cycocel and Bettaquat, two commonly used growth regulators, or urea, a frequently used f o l i a r feed had any b e n e f i c i a l e f fects i n these t r i a l s . In fact the authors describe a s i g n i f i c a n t y i e l d decrease (9.3%) in response to 40 kg ha of urea (Miers and Perry, 1986). Temple and Bomke (University of B r i t i s h Columbia, personal communication, 1987) and Dobb ( B r i t i s h Columbia Ministry of Agriculture and Fisheries, personal communication, 1986) reported s t a t i s t i c a l l y s i g n i f i c a n t increases i n y i e l d of beans, barley, wheat and canola treated - i with seaweed spray (1 to 2 1 ha ) made from Macrocystis  i n t e g r i f o l i a . Recently Aldworth and van Staden (1987) published results from an experiment i n which potted marigold (Tagetes patula) seedlings were treated once with 2 ml of 1:1, 1:2 or 1:4 d i l u t i o n s of Kelpak at the four leaf stage. After six weeks growth, s i g n i f i c a n t increases i n the root and shoot fresh and dry mass, number of leaves, flowers and flower buds, and stem diameter were measured: a l l were approximately double the control values. This paper also reported a s i g n i f i c a n t increase i n cabbage root and shoot fresh and dry weights resulted from a f i v e minute immersion of the roots i n 1:500 seaweed d i l u t i o n p r i o r to planting. In another experiment from the same laboratory using groundnut (Arachis hypogaea) f o l i a r seaweed was applied at 3 or 3 and 8 weeks from emergence (20 ml, 1:400 d i l u t i o n ) . Results indicate a 35% increase i n y i e l d from a single application, and a 65% increase from the double application. There was also a large increase i n the number of large seeds (Featonby-Smith and van Staden, 1987). 1.4.2 Tissue Composition: It has been demonstrated that seaweed sprays can a l t e r s i g n i f i c a n t l y the chemical and physical composition of the plants to which they are applied. 20 Aitken and Senn (1964) noted s l i g h t differences i n the pH, t o t a l a c i d i t y , soluble so l i d s and moisture content of treated and untreated peaches. Bunch grapes showed no differences i n soluble solids or t o t a l a c i d i t y , while melons from plants treated with seaweed spray had a 2-3% higher 2+ 2+ sugar content and increased Mg , N, and Ca content. Tobacco plants sprayed with seaweed showed an increase i n a l k a l o i d content accompanied by a decrease in t o t a l N. More recently, several papers have been published concerning the e f f e c t of seaweed sprays on the a l k a l o i d content of Duboisia; these alkaloids are used i n drug manufacture. Luanratana and G r i f f i n (1980a,b, 1982) showed that Maxicrop increased the t o t a l a l k a l o i d content of leaves of hydroponically-grown plants, with a higher dose (1%) y i e l d i n g increases in hyoscine content and a lower dose (0.5%) increasing the hyoscyamine concentration. In the f i e l d , the t o t a l a l k a l o i d increase as a result of Maxicrop application was i n s i g n i f i c a n t (5.8%); however the 16% increase i n hyoscine over controls was s i g n i f i c a n t . The authors suggest that the seaweed spray may have delayed the normal seasonal decline i n a l k a l o i d content, and indeed subsequent experiments demonstrated that the May to September decline i n hyoscine content did not occur i n seaweed-treated plants (Luanratana and G r i f f i n ,1982). Stephenson (1974) reported increased concentrations of 21 2+ 2 + protein (3.8-5.0%), N, P, Ca , and Zn i n grass after a -1 single application of fi v e pints acre of Algistim. Goh and Whitton (1975) reported a soil-type dependent increase i n the uptake and content of CI, S, Na, and K of white clover plants, followed by a decrease to control l e v e l s as the plants aged. Featonby-Smith and van Staden (1987) found that Kelpak had no eff e c t on the N or moisture content of wheat, but increased the chlorophyll content of leaves of Beta vulgaris (Featonby-Smith and van Staden, 1983). Blunden 2+ (1972) found increases i n Mn uptake by banana leaves at 0.5 -1 and 1.0 gal. acre , but observed decreases at 0.75 and 1.25 -1 2+ 2+ gal acre . No effects on N, P, K, Ca , or Mg were detected. Significance levels were not reported. Nelson and van Staden (1984) obtained s i g n i f i c a n t reductions i n the N content (12%) and increases i n the P content (20%) of leaves of "nutrient-stressed" cucumbers i n the greenhouse. Seaweed concentrate applied to several sugar beet v a r i e t i e s resulted in s i g n i f i c a n t l y higher root sugar content (4.4%), while amino-N and K content of the juice was s i g n i f i c a n t l y reduced. Several published papers describe changes i n the tissue composition of seaweed-treated f r u i t trees. Roder et, a l . (1985) recorded s i g n i f i c a n t increases i n peach f o l i a r P (40%), Mn 2 + (31%), and Z n 2 + (29%). Levels of N, K, C a 2 + , Mg 2 +, -1 Fe, and Cu were not affected by the spray (4 X 127 1 ha applications). Povolny (1969), sprayed 'Cox Rennet' apple trees with 0.8% A l g i f e r t 2-4 weeks p r i o r to harvesting and found no changes i n soluble refractometric dry matter, t o t a l a c i d i t y or sugar content of harvested f r u i t . De V i l l i e r s ej: a l . (1983) sprayed seaweed preparations on peach and apple trees, as well as grape vines, and measured leaf N, P, K, 2+ 2 + Ca , Mg , Mn, Cu, B, and Zn. They recorded s i g n i f i c a n t increases i n the P content (29%) of 'Van Riebeek' peach leaves, and a 16% increase in the Mn content of 'Starking' apple leaves. There was no ef f e c t on 'Golden Delicious' apple or table grape f o l i a r Mn content, or table grape f r u i t g u a l i t y . Senn and Kingmann (1978), however, reported that grapes sprayed weekly from bloom to harvest with seaweed possessed a s i g n i f i c a n t l y higher soluble s o l i d content than controls, while pH was not affected. De V i l l i e r s et a l . 2+ 2 + (1983) could detect no changes i n the Ca , Mg , or P m the f r u i t of seaweed treated apple trees, and no alterations i n 2 + leaf chlorophyll l e v e l s . The same paper reported that Ca isotope uptake by bean plants was increased by seaweed application, and Zn uptake by apple seedlings was decreased. Differences i n ef f e c t arose both from the seaweed product used and the concentration applied. Extensive experiments at Clemson University revealed changes i n the tissue composition of several crops i n response to weekly spray applications of Ascophyllum nodosum preparations. Soybeans responded with s i g n i f i c a n t increases i n moisture (3%), acid (52%), and protein (7%) contents, and a s i g n i f i c a n t reduction i n o i l (7%) content. Tomato f r u i t s from sprayed plants had s t a t i s t i c a l l y higher soluble solids (7%) after processing. The f i b r e content of treated okra was unaffected, nor were the pH, t i t r a t a b l e acids, or soluble so l i d s of sweet corn or southern pea. The seaweed spray had 2+ 2 + no s i g n i f i c a n t e f f e c t on concentrations of K, Ca , Mg , Zn, Fe, Mn, or P i n leaves of b r o c c o l i (Brassica oleracea var. i t a l i c a ) , turnip (Brassica rapa), cauliflower or cabbage (Senn and Kingman, 1978). Kotze and Joubert (1980), however, 2 + reported a decrease in the levels of cabbage f o l i a r Ca and Cu after seaweed treatment. They also found increases i n the 2+ 2 + uptake of Ca , Mg , K, and P by rye plants treated with f o l i a r seaweed. Senn and Kingman (1978) also report effects on pea, tomato and okra i n which treatment i s followed by increased 2 + calcium (500%) and Mg (50%) i n treated pea plants; i n 2 + tomato plants, however, Mg was decreased by 20%. Zn concentrations were increased i n okra (100%) and tomato (300%), but decreased in peas (-300%). Seaweed sprays gave large increases i n the Fe concentrations of a l l three test species; okra by 400%, peas by 300%, and tomato by 425%. Increased Mn content (300%) occurred i n peas only. P leve l s were doubled i n okra, unaffected i n tomato and reduced (-26%) in peas. Significance levels were not reported. Further experiments at Clemson i n 1977 (Senn and Kingman, 1978) showed a 4% increase i n soluble s o l i d s but no eff e c t on pH or t i t r a t a b l e acids of seaweed f o l i a r sprays (1 gal. acre i ) applied to tomato plants.at emergence, mid-growth and f r u i t set. Neither did similar treatments applied to lima beans (Phaseolus limensis) aff e c t these parameters. Dwelle and Hurley (1984) saw no differences i n s p e c i f i c gravity among potatoes of various clones and c u l t i v a r s as a resul t of spray treatment (Cytex) at d i f f e r e n t times and rates. Recent f i e l d t r i a l s with beans showed s i g n i f i c a n t changes in elemental concentration and uptake rates when control levels were compared to sprayed levels (Temple et aJU, 1986) . Seaweed application resulted i n s i g n i f i c a n t l y reduced K and 2 + Mg levels i n pods across a l l treatment sprays, while uptake of N, P, K, and Cu were increased. No eff e c t on leaf n u t r i t i o n or status of other essential elements was observed. A subsequent experiment showed a s i g n i f i c a n t decrease i n bean 2+ 2 + pod Zn concentration, and decreased uptake of K, Ca , Mg , and Fe by pods. Again no differences i n le a f composition between the control and spray treatments were evident. In a companion greenhouse study a sim i l a r trend existed; i . e . reduced uptake but increased concentrations, primarily i n the f r u i t , as opposed to the foliage of treated plants. An inte r a c t i o n of t h i s e f f e c t with s o i l water potential was also established Featonby-Smith and van Staden (1987) obtained s i g n i f i c a n t l y higher (12-18%) protein content i n small and intermediate-sized groundnut seeds from plants sprayed twice with 20 ml of 1:400 Kelpak. Differences i n large seeds, and t o t a l per cent seed protien were not s i g n i f i c a n t . No differences i n l i p i d content between control and seaweed treated plants were measured. F i n a l l y , Featonby-Smith and van Staden (1984) measured the e f f e c t s of seaweed f o l i a r sprays of Kelpak on the endogenous cytokinin content of Phaseolus vu l g a r i s . Their results showed that t h i s treatment increased the leve l s of -1 -1 cytokinin i n the plants from 75 ng g to 675 ng g ki n e t i n equivalents. It also increased the chlorophyll content by 40%. In a si m i l a r study with swiss chard (Beta vulgaris v a r . c i c l a ) , Featonby-Smith and van Staden (1983) found that the c y t o k i n i n - l i k e a c t i v i t y of root extracts of treated plants was greater than i n control plants, and the converse in l eaf cytokinin a c t i v i t y . Total c y t o k i n i n - l i k e a c t i v i t y was lower i n seaweed-treated plants than i n controls. 1.4.3 Development and Dry Matter P a r t i t i o n i n g : In addition to the germination and emergence effects described above (Section 1.3), several published reports indicate that, i n some situations, developmental processes such as the rate and d e g r e e o f f l o w e r a n d f r u i t p r o d u c t i o n , a n a t o m i c a l c h a n g e s , a n d d r y m a t t e r a l l o c a t i o n c a n be a l t e r e d b y a p p l i c a t i o n o f seawee d f o l i a r s p r a y s . F u r t h e r m o r e , s i g n i f i c a n t i n t e r a c t i o n s b e t w e e n r e s p o n s e a n d t i m i n g o f s p r a y a p p l i c a t i o n ( i . e . d e v e l o p m e n t a l s t a g e ) h a v e b e e n r e p o r t e d . A commonly r e p o r t e d d e v e l o p m e n t a l e f f e c t r e s u l t i n g f r o m s e a w e e d f o l i a r a p p l i c a t i o n i s t h a t o b s e r v e d f o r d r y m a t t e r a l l o c a t i o n . S e v e r a l a u t h o r s h a v e r e p o r t e d s h i f t s i n t h e y i e l d component d i s t r i b u t i o n t h a t i n d i c a t e d i s p r o p o r t i o n a t e r o o t , s e e d a n d f r u i t g r o w t h c a n o c c u r i n r e s o n s e t o se a w e e d f o l i a r s p r a y s . B l u n d e n a n d W i l d g o o s e (1977) d e s c r i b e i n c r e a s e s i n r o o t mass, l a t e r a l r o o t d e v e l o p m e n t and u n i f o r m i t y , as w e l l a s i n c r e a s e d s t e m l e n g t h i n p o t a t o as a r e s u l t o f f o l i a r l y a p p l i e d s e aweed. D w e l l e a n d H u r l e y (1984) f o u n d a v a r i e t a l i n t e r a c t i o n , w i t h no r e s p o n s e f r o m ' R u s s e t B u r b a n k ' a n d e r r a t i c r e s p o n s e s f r o m 'Lehmi R u s s e t ' s p r a y e d w i t h Cytex. They i n d i c a t e d t h a t t h e t i m i n g o f a p p l i c a t i o n was i m p o r t a n t f o r y i e l d i n c r e a s e s t o o c c u r ; t r e a t m e n t a t t h e o n s e t o f t u b e r i n i t i a t i o n was most e f f e c t i v e . L a n g a n d L a n g v i l l e (1984) o b s e r v e d no a l t e r a t i o n i n t u b e r number, b u t i n c r e a s e s i n i n d i v i d u a l t u b e r s i z e when t h e c a n o p y o f p o t a t o v a r i e t i e s was s p r a y e d w i t h Cytex. They a l s o p o i n t e d o u t an i n c r e a s e i n t h e o c c u r r e n c e o f d e f e c t i v e p o t a t o e s ( c r a c k s , s u n b u r n ) f r o m t h i s t r e a t m e n t , r e f l e c t i n g i n c r e a s e d c o m p e t i t i o n w i t h i n t h e h i l l 27 and displacement of upper tubers to the surface. Nelson and van Staden (1984) obtained s i g n i f i c a n t l y increased root/shoot ra t i o s from cucumber plants sprayed weekly with seaweed sprays. They also noted an i n i t i a l i n h i b i t i o n of f r u i t growth, yet f a i l e d to report ultimate f r u i t y i e l d . A similar increase was recorded by Featonby-Smith and van Staden (1984), who observed an o v e r a l l 24% increase i n dry mass of bean plants, with a 20% increase i n the root/shoot r a t i o . Root growth was also stimulated i n tomato plants grown in nematode-infested s o i l , however root/shoot ra t i o s were unaffected (Featonby-Smith and van Staden, 1983). Subsequent experiments from the same laboratory used tomato roots cultured i n v i t r o to demonstrate that d i l u t e solutions of seaweed preparation stimulated both root elongation and development of l a t e r a l s . Concentrations higher than 1:100 i n h i b i t e d these ef f e c t s , while more d i l u t e than 1:1000 had no ef f e c t (Finnie and van Staden, 1985). Large increases i n root/shoot r a t i o were described by Kotze and Joubert (1980) for cabbage sprayed with 1:330, 1:500 and 1:1000 d i l u t i o n s of seaweed. Shoot y i e l d was unaffected, but root weights increased 100%, 50% and 30% respectively. In p a r a l l e l experiments with rye, root growth was doubled by seaweed f o l i a r application, while shoot growth increased 50-85%, r e s u l t i n g i n a higher root/shoot r a t i o than controls. 28 Nelson and van Staden (1985) reported considerable increases i n wheat culm diameter (100%) and length (13%) following s o i l treatment with Kelpak. Dilute seaweed extract applied as a f o l i a r spray, as well as as a s o i l drench increased the o v e r a l l size of individual vascular bundles i n both the inner and outer vascular rings. Increased diameters were attributed to increases in c e l l size rather than c e l l number. In addition to these stem effects, increases i n the number of spikelets per ear, grain weight per ear, and chlorophyll retention were described. More information on e f f e c t s of seaweed sprays on reproductive structures of barley was given i n Featonby-Smith and van Staden (1987). The number of ears increased by 41% after application of a spray of 1:250 Kelpak. This was the result of increased t i l l e r i n g rather than more advanced head development on treated plants. The number of spikelets per ear was unchanged by the spray, i n contrast to previous results with barley from the same laboratory (Nelson and van Staden, 1985), but more f e r t i l e spikelets were counted on seaweed treated plants (77-82%) than on controls (58%). This combined increase i n t i l l e r i n g and per cent f e r t i l e spikelets produced the increased y i e l d . Ketring and Shubert (1981) were unable to obtain consistent e ffects on reproductive structures of peanut plants treated with Cytex. In only one of several 29 experiments did they note an increase in t o t a l pegs. A trend towards increased pod and seed number and weight was mentioned, but was not s t a t i s t i c a l l y s i g n i f i c a n t . Further information on the response of potted groundnuts to seaweed application was given by Featonby-Smith and van Staden (1987). Two seaweed product applications produced a s i g n i f i c a n t 36% increase i n the number of single-pod f r u i t s produced, with no change i n the r a t i o of peg/fruit set. The ra t i o of s h e l l mass to seed mass was decreased by the seaweed spray, i n d i c a t i n g a s h i f t i n reproductive dry matter a l l o c a t i o n . Temple et al.. (1986) reported disproportionate increases in shoot (19%) and f r u i t (30%) yields of field-grown Phaseolus, in d i c a t i n g a s h i f t i n dry matter p a r t i t i o n i n g from the vegetative to the reproductive structures i n response to seaweed spray f o l i a r application. Subsequent greenhouse experiments demonstrated reduced leaf area r a t i o despite increases i n the shoot, leaf and stem weights, i n d i c a t i n g a change i n the composition and/or structure of the leaves i . e . although leaf area was unchanged, leaf dry weight was increased. This r e f l e c t e d a change to thicker, more dense leaves. Increased photosynthetic c a p a b i l i t y was also implied, since more dry matter was accumulated by the same leaf area. Featonby-Smith and van Staden (1983) examined growth 30 effects on swiss chard of f o l i a r application of seaweed extracts. They found increased numbers of leaves and leaf area per plant. Chlorophyll content was also higher i n treated plants. Although root growth was stimulated, shoot responses were also evident and no change was observed i n root/shoot r a t i o . Corn plants treated with seaweed extract by Blunden (1977), i n an experiment under undefined conditions, were t a l l e r and had broader, greener leaves and thicker stalks. Furthermore, corn from treated plants tasseled and "ripened e a r l i e r . Similar accelerated development occurred on treated banana plants, which shot 8.5 weeks e a r l i e r than control plants. In contrast, Povolny (1976) reported a retardation of ripening ( i . e . increased hardness) of peaches and apricots (Prunus armeniaca), as well as apples (Povolny, 1969) and tomatoes (Povolny, 1976) from sprayed plants. Considerable a l t e r a t i o n in growth of marigolds treated with seaweed extract as a root drench and f o l i a r spray was reported by Aldworth and van Staden (1987). Both methods of application, s i g n i f i c a n t l y increased root and shoot dry mass, stem diameter, and number of leaves, flowers and flower buds. The increase i n number of shoots per plant point towards a reduction i n apical dominance as a result of seaweed spray. The same paper reported an increase in cabbage stem length and diameter r e s u l t i n g from a f i v e minute root dip i n 1:500 d i l u t i o n of Kelpak. A similar e f f e c t on apical dominance and bud break i n greenhouse roses (Rosa canina) was recounted by Raviv (198 6). He found that 14-47% more bottom breaks ( i . e . renewal shoots) were formed on roses of three v a r i e t i e s sprayed once with 1% Seamac 600. More plants produced bottom breaks, with more bottom breaks per plant. The author suggested a seasonal interaction, with the e f f i c a c y of the seaweed spray at increasing bottom breaks related to the penetration of l i g h t to the base of the plants: the more l i g h t penetration, the more pronounced was the e f f e c t . 1.4.4 Storage Quality: Seaweed treatment of f r u i t s and vegetables pre- or post-harvest has been shown to a l t e r t h e i r storage c h a r a c t e r i s t i c s . In many of the papers c i t e d below, increased shelf l i f e seems to be the result of increased hardness, or retardation of ripening on the plant (e.g. Povolny 1969,1972,1976). Skelton and Senn (1969) advanced the opinion that although control f r u i t s were s l i g h t l y softer, t h i s difference could not account for the differences in s h e l f l i f e . Furthermore, seaweed treatment, e s p e c i a l l y post-harvest, can accelerate ripening (Blunden et a l . , 1978). Driggers and Marucci (1964) noted reduced incidence of mould and monilioses on sprayed f r u i t , i n dicating possible a n t i b i o t i c or disease resistance mechanisms of action. Pre-harvest Treatment: Senn and Skelton (1966) reported that the s h e l f l i f e of 'Sullivan Elberta' peaches, as measured by the percent marketable f r u i t s following storage, was increased by pre-harvest spraying of the trees. Similar results were obtained with 'Rio-Oso-Gem' peaches. Skelton and Senn (1969) noted that, although the f r u i t s from sprayed trees were s l i g h t l y firmer at harvest, the change was not enough to account for the improved storage c h a r a c t e r i s t i c s . Of peaches sprayed pre-harvest with seaweed extract by Driggers and Marucci (1964), 14.7% had rotted after 16 days storage, compared to 32.2% of control f r u i t s . In a similar study over a four year period Skelton and Senn (1969) recorded a reduction i n losses from 50% (control) to 20% (pre-harvest seaweed spray) of 'Blake' peaches stored for three weeks. Greatest increases i n f r u i t shelf l i f e were noted for f r u i t trees sprayed with seaweed early i n the season. Another investigation of peach and apricot shelf l i f e was performed over a three year period by Povolny (1972). Applying 0.4 or 0.5% A l g i f e r t at a rate of 10 1 _ i tree increased the hardness of peaches by a factor of four over control f r u i t s , r e s u l t i n g i n a 35% decrease i n storage losses. S i m i l a r l y apricot hardness was increased two-fold, decreasing storage losses by 13-35%, depending on variety. The authors noted that the ripening of treated f r u i t s was delayed. Several studies have been car r i e d out to assess the effect of pre-harvest seaweed spray application on shelf l i f e of apples. Povolny (1969) sprayed three apple v a r i e t i e s with 0.8% A l g i f e r t four and two weeks p r i o r to harvest. 'Cox rennet' apple shelf l i f e was most affected, with only 4.3% of the apples from treated trees rotted after 30 days storage, compared to 39.2% of controls. Control apples were ri p e r when picked, hence the 60% harder pulp of treated f r u i t s . 'Matcino' apples showed a lesser response, with 8% harder pulp and 10.2% of apples from seaweed treated trees rotted after 32 days storage, compared to 21.3% of control f r u i t . No e f f e c t on storage quality of 'Golparmane' apples was measured i n response to seaweed sprays. More recently, D e V i l l i e r s et a l . (1983) found no ef f e c t on f r u i t quality resulted from spraying 'Golden Delicious' or 'Starking' apple trees with either of two seaweed products. Neither was any eff e c t on storage quality of grapes from treated vines observed. Povolny (197 6) reported that 0.5% A l g i f e r t sprayed onto tomato plants at two week interv a l s resulted i n an 18-35% increase i n f r u i t hardness, accompanied by 23-45% decreases in f r u i t loss after four weeks storage. With cucumbers from sprayed plants (0.04% A l g i f e r t , weekly) storage l i f e was increased by 14-21 days (Povolny, 1968). F i n a l l y Blunden 34 (1972) claimed 100% marketability of peppers from plants receiving pre-harvest sprays with seaweed preparations following eight days storage, compared to complete unmarketability of f r u i t s from control plants. Methods of quantification, experimental design and significance levels were not given. Post-harvest Treatment: A single study reports the effects of soaking harvested f r u i t i n various concentrations of SM-3 and Marinure. No eff e c t was noted on storage quality of eggplants (Solanum melonciena), avocadoes (Persea americana) or pears. Bananas and mangoes (Manqifera indica) both responded with accelerated ripening when soaked i n d i l u t e seaweed solutions. Peppers immersed i n d i l u t e seaweed extract had reduced rates of reddening, while limes (Citrus  aurantifolia) so treated had reduced rates of de-greening i . e . delayed ripening (Blunden, 1978). 1.4.5 Disease and Pest Resistance: Senn and Kingman (1978) reported that i n the course of an experiment on the effects of seaweed spray on growth of melons, they had observed a lowered incidence of fungal pathogens i n treated plants. These casual observations were subsequently confirmed i n f i e l d t r i a l s . S imilarly, Stephenson (1966) reported that when turnips were grown i n conditions favourable to the development of 35 powdery mildew 85% of the control leaf area became infested, whereas only 15% of the l e a f area of seaweed sprayed plants was affected. He also observed y i e l d increases similar to those obtained by applying a commercial fungicide i n conjunction with the seaweed extract. The same publication describes strawberry plants watered weekly overhead with 1:120 d i l u t i o n of Maxicrop. Botrytis on ripe berries decreased to about 20% of the l e v e l on untreated plants. This r e s u l t was duplicated i n three successive t r i a l s . Stephenson (1966) also reported control of tomato damping-off in which 95% of treated (1:20 d i l u t i o n of Maxicrop) seedlings reached the four l e a f stage, compared to only 45% of control seedlings. Darrah and H a l l (197 6) observed that granular seaweed applied to the s o i l reduced the populaton of Fusarium by 48%, to a l e v e l comparable to that i n Benomyl-treated plots; l i q u e f i e d seaweed had no e f f e c t . Abetz and Young (1983) make vague mention of a reduction in the percentage of "diseased or failed-to-heart" lettuce following seaweed spray and s o i l application. Povolny (1969) reported a reduction i n brown speckling on 'Cox Rennet' apples receiving pre-harvest f o l i a r seaweed extract applications. Driggers and Marucci (1964) and Aitken and Senn (1965) both obtained p a r t i a l control of brown rot i n peaches through seaweed application. As mentioned above Driggers and Marucci (1964) state that the incidence of pathogens was reduced on harvested f r u i t . Several authors have reported that seaweeds can have a protective e f f e c t against plant pests. Stephenson (1966) recorded several experiments i n which insect or mite infestations were reduced by l i q u e f i e d seaweed application. He described the d i s t r i b u t i o n of winged aphids on sprayed (20%) versus unsprayed (83% sic) black bean leaves i n feeding preference t e s t s . Apterae reproduced less, he stated, on treated compared with untreated leaves primarily as a result of "unsettled behavior". Peach potato aphids were affected in a s i m i l a r ( i . e . non-insecticidal) manner. Populations of red spider mites, both i n orchard f i e l d tests and on chrysanthemums i n the greenhouse, were s i g n i f i c a n t l y reduced by seaweed extract f o l i a r treatment at two week i n t e r v a l s . Although Darrah and Hall (1976) found that f o l i a r application of l i q u i f i e d seaweed had no e f f e c t on Fusarium populations, granular seaweed was e f f e c t i v e i n reducing them. Associated with t h i s e f f e c t was a concurrent 98% reduction i n the nematode population after three years. In 1983 Featohby-Smith and van Staden published more information on the e f f e c t of seaweed extract treatment on nematode-infested plants. They found that a single s o i l drench, or repeated f o l i a r application of seaweed extract to tomato plants resulted i n root, shoot and f r u i t y i e l d increases. Nematode populations i n s o i l flushed with seaweed extract were increased, yet the number of nematodes established i n the root systems was reduced. P r i o r to t h i s , Morgan and Tarjan (1981) had reported p a r t i a l control of one of three sting nematode species by two seaweed products, Maxicrop and Sea-Born. B'chir et ajL. (1983) also reduced s o i l and cucumber root i n f e s t a t i o n by Meloidoayne when seaweed extract was applied with Carbofuran and Arthrobotrys, but not by either nematicide or fungus alone. 1.5 MECHANISM OF ACTION OF SEAWEED FOLIAR SPRAYS Two hypotheses have been proposed to account for the diverse effects of seaweed f o l i a r sprays'reviewed i n the previous section. 1.5.1 N u t r i t i o n a l Hypothesis: It has been proposed that the crop response to f o l i a r seaweed application was due to the nutrient content of the spray. It i s well known that seaweeds are r i c h in both micronutrients and vitamins that could contribute to the n u t r i t i o n of crop plants. Table 2 l i s t s the elemental content of several seaweed products and t y p i c a l application rates i n d i c a t i n g the amount of each element applied, and compares t h i s to the elemental content of sugarbeet. It i s evident from t h i s table that at common 38 KELPAK Af. integrifolia CROP AMOUNT % OF AMOUNT % OF CONTENT APPLIED CROP APPLIED CROP ELEMENT (kg ha - 1) (g ha" 1) CONTENT (g ha - 1) CONTENT N 126 42 0.03 300 0.2 P 66 5.4 0.008 6.0 0.009 K 241 43 0.02 60 0.02 Ca 63 2.8 0.004 7.2 0.01 Mg 25 1.2 0.005 4.6 0.02 Fe 1.9 0.0007 0.00004 0.24 0.01 Cu 0.44 0.0005 0.001 0.005 0.01 Mn 0.52 0.0004 0.00008 0.009 0.002 Zn 0.19 1.32 .007 0.07 0.04 TABLE 2 Elemental Content of Beet (Beta vulgaris) Crop Compared to Elemental Content of F o l i a r l y Applied Seaweed Preparations Elemental content of crop i s based on data from Draycott, (1985). Elemental content of seaweed preparations i s based on data from Abetz (1980), and Temple et al. (1986), and assumes 3 applications of 2 1 ha 39 application rates, seaweed sprays contribute n e g l i g i b l e elemental n u t r i t i o n to the crops. In no case does the application of any element exceed 0.7% of i t ' s corresponding quantity i n the crop. Furthermore, experiments with radio-l a b e l l e d elements indicate that 192 hours after application as l i t t l e as 15% of elements applied to foliage are absorbed by bean leaves (Fergoni, 1985). Several other factors argue against a n u t r i t i o n a l mechanism for seaweed spray a c t i v i t y . Although there are few published reports of d i r e c t investigations of the interaction of s o i l f e r t i l i t y with seaweed f o l i a r application, several inferences can be drawn from the elemental analyses described above. In cases where the application of seaweed has s i g n i f i c a n t l y altered the elemental composition of the crop, the alterations do not correspond to the elemental composition of the seaweed. This difference suggests that uptake e f f i c i e n c y or a v a i l a b i l i t y of s o i l borne nutrients may have been altered by the seaweed. In cases where root mass has been enlarged r e l a t i v e to the shoot mass, t h i s could be duepartially to increased interception of nutrients. Several studies (e.g. Darrah and H a l l , 1976; Featonby-Smith and van Staden, 1983, 1984; Kingman and Senn, 1978,) reported no loss of y i e l d i n several crops f e r t i l i z e d at l e v e l s below the recommended rates when f e r t i l i z a t i o n was 40 accompanied by f o l i a r seaweed extract application. Y i e l d increases, however, were greater i n response to seaweed extract application to plants receiving less f e r t i l i z e r (111%), than to plants receiving more f e r t i l i z e r (36%) (Featonby-Smith and van Staden, 1983, 1984). Kingman and Senn (1978) attributed such y i e l d responses to the e f f i c i e n t absorption of the chelated micronutrients that seaweed provides, but acknowledged that they observed "plant responses which could not be explained by the chemical analysis of the seaweed". Kotze and Joubert (1980) recounted greater e f f e c t s of seaweed extracts on root growth of both cabbage and rye at high f e r t i l i z e r levels than at lower rates. They stressed, therefore, that seaweed applications could not be used to improve poor growth r e s u l t i n g from n u t r i t i o n a l d e f i c i e n c i e s . In comparisons of the elemental analyses of control plants with plants sprayed with seaweed extract, no nutrient d e f i c i e n c i e s as defined by low tissue levels of s p e c i f i c elements have been corrected or elevated by seaweed application. As D e v i l l i e r s et a l . (1983) pointed out i n t h e i r experiments with apples and grapes, elemental content 2+ 2 + of Ca , P, Mn and Zn was increased at some seaweed application rates, but t h i s e f f e c t was not " p r a c t i c a l l y s i g n i f i c a n t " because these elements were already present i n adequate amounts. In cases where analysis showed d e f i c i e n c i e s ( i . e . boron), the deficiency was not corrected by seaweed application. A f i n a l i n d i c a t i o n that crop growth responses are not d i r e c t l y or solely n u t r i t i o n a l comes from a paper by Finnie and van Staden (1985). They demonstrated complete loss of growth-promoting a c t i v i t y when the seaweed preparation they used was ashed before application. This observation indicated that some organic constituent, rather than the elemental composition of the seaweed, was l i k e l y responsible for i t s a c t i v i t y . The r e j e c t i o n of the nutrient hypothesis to explain seaweed spray a c t i v i t y , except perhaps i n cases of extremely marginal micronutrient d e f i c i e n c i e s , leads to speculation about organic constituents of the seaweed that could a f f e c t plant growth so broadly and so s i g n i f i c a n t l y at the application rates used. 1.5.2 Phytohormonal Hypothesis: The demonstration by Finnie and van Staden (1985) that the growth-promoting e f f e c t s of seaweed extracts were lo s t upon ashing, combined with the effectiveness of low application rates and the diverse e f f e c t s of these f o l i a r treatments reinforced the proposition that organic compounds, s p e c i f i c a l l y phytohormones or growth regulators i n seaweed, were responsible for t h e i r a c t i v i t y (Booth, 1966). It i s well known that seaweeds contain compounds wi t h c y t o k i n i n - l i k e , a u x i n - l i k e and g i b b e r e l l i n -1 . . . . . l i k e a c t i v i t y , and auxin and c y t o k i n i n have been unequivocally i d e n t i f i e d from s e v e r a l a l g a l sources (e.g. Tay et a l . , 1 9 8 5 ) . G i b b e r e l l i n s : G i b b e r e l l i n - l i k e a c t i v i t y has been reported i n e x t r a c t s of s e v e r a l marine algae. Radley (1956) f i r s t detected g i b b e r e l l i n - l i k e a c t i v i t y from Fucus v e s i c u l o s u s . Bentley (1960) detected g i b b e r e l l i n - l i k e a c t i v i t y from phytoplanktonic sources, and P r o v a s o l i (1958) reported phytohormonal a c t i v i t y i n seawater. Bergland (1969) found that seawater from the Ascophyllum-Fucus l i t t o r a l zone was u s e f u l f o r a l g a l c u l t u r e , probably because i t contained n u t r i t i o n a l and growth r e g u l a t o r y f a c t o r s of macrophytic o r i g i n . The g i b b e r e l l i n a c t i v i t y found by Radley (1956,1961) i n the brown macroalga Fucus v e s i c u l o s u s was v e r i f i e d by Mowat (1964) , who judged the g i b b e r e l l i n content at about 0 . 1 - 1 0 ug -1 . . . kg f r e s h weight, based on the b i o l o g i c a l a c t i v i t y of e x t r a c t s compared to GA^ standards. Later, Fucus s p i r a l i s was a l s o found to contain g i b b e r e l l i n - l i k e substances (Mowat, 1965). Kato et a l . (1962) were unable to detect g i b b e r e l l i n -l i k e a c t i v i t y from the kelp Macrocystis p y r i f e r a , however Radley (1978) showed g i b b e r e l l i n - l i k e a c t i v i t y from the i F o l l o w i n g the convention suggested by Taylor and Wilkinson (1977), substances w i t h b i o l o g i c a l a c t i v i t y i n recognized phytohormonal bioassays, but wit h unknown chemical s t r u c t u r e s w i l l be s u f f i x e d w i t h " - l i k e " . 43 closely related. Nereocystis luetkeana. Murakami (1965) demonstrated the presence of g i b b e r e l l i n -l i k e substances i n Rhodophytes, and Jennings (1968) estimated the g i b b e r e l l i n - l i k e a c t i v i t y of Ecklonia (Phaeophyta) and - i Enteromorpha (Chlorophyta) at about 100 ug kg g i b b e r e l l i c acid (GA.j) equivalents fresh weight based on extraction, chromatography and bioassay. He also demonstrated that seaweed in culture responded to extracts and chromatographic fractions obtained by methods designed for g i b b e r e l l i n i s o l a t i o n in the same way i t did to synthetic g i b b e r e l l i n . Further, he described a s p e c i f i c g i b b e r e l l i n antagonist from the seaweed. A l a t e r publication (Jennings, 1971) showed that CCC (chlormequat chloride) i n h i b i t i o n of Ecklonia growth in culture was overcome by addition of GA^ to the medium. Since these early investigations, dozens of a l g a l species have been shown to contain substances active i n g i b b e r e l l i n bioassays, and many of these species respond to exogenous GA in culture as well (Augier, 1976/ Taylor and Wilkinson, 1977) . Taken together, the following facts strongly imply a regulatory role for g i b b e r e l l i n s i n algae similar to that i n higher plants: 1) widespread endogenous occurrence i n algae/ 2) widespread and appropriate response to exogenous application/ 3) presence of GA-specific antagonists, i . e . the potential for regulation through a balance or r a t i o of growth stimulators and i n h i b i t o r s ; and 4) reversal of the effects of a s p e c i f i c GA i n h i b i t o r (CCC) by algal extracts. Despite such strong circumstantial evidence i n favor of a regulatory role for g i b b e r e l l i n s i n algae, rigorous chemical i d e n t i f i c a t i o n of an a l g a l g i b b e r e l l i n has yet to be accomplished. With respect to commercially available seaweed sprays, Williams e i a l . (1981) tested three products for the presence of g i b b e r e l l i n - l i k e a c t i v i t y . They found that while fresh extracts contained g i b b e r e l l i n - l i k e a c t i v i t y corresponding to -1 . . . . 0.03 to 18.4 mg 1 GA^ equivalents, t h i s a c t i v i t y declined u n t i l by f i v e months after manufacture, a l l g i b b e r e l l i n - l i k e a c t i v i t y had disappeared. Kingman (1975) also described g i b b e r e l l i n - l i k e a c t i v i t y i n extracts of Ascophyllum nodosum from which a g r i c u l t u r a l f o l i a r sprays were made. Because of the general i n s t a b i l i t y of g i b b e r e l l i n s i n aqueous solution (Hubbick and Reid, 1982), the extremes of temperature and pH to which alg a l tissues are subjected during manufacture, (e.g. Sanderson and Jameson, 1986) and the decline i n g i b b e r e l l i n - l i k e a c t i v i t y of various seaweed extracts with time (Williams et a l . , 1981), i t i s u n l i k e l y that the g i b b e r e l l i n content of seaweed sprays could explain t h e i r e f f i c a c y . According to Graebe and Ropers (1978), the production of g i b b e r e l l i n decomposition products which are inactive i n bioassays i s greatly catalyzed by acids and bases. Autoclaving GA^ results i n 98-99% destruction. Since many seaweed sprays are boiled i n alkaline solution during manufacture, i t i s unlikely that s i g n i f i c a n t g i b b e r e l l i n - l i k e a c t i v i t y would p e r s i s t . Even i n the case of freshly manufactured seaweed spray -1 containing up to 18 mg 1 GA^ equivalents of g i b b e r e l l i n -l i k e a c t i v i t y , the recommended d i l u t i o n rates of 100- to 500-f o l d reduce the g i b b e r e l l i n - l i k e a c t i v i t y of the applied -1 spray to about 180 ug 1 . Most a g r i c u l t u r a l applications c a l l for about 1-1000 mg l - 1 , t y p i c a l l y 20-50 mg l - 1 (Considine, 1983), thus seaweed would be unable to supply enough g i b b e r e l l i n - l i k e a c t i v i t y for a g r i c u l t u r a l use. In contrast, the barley endosperm bioassay can detect as l i t t l e as 100 ug l " 1 GA3 (Greabe and Roper, 1978). There i s therefore enough g i b b e r e l l i n in some freshly-prepared seaweed products to evoke physiological responses i n s p e c i f i c tissues and organs under conditions s p e c i f i c a l l y designed to maximize the g i b b e r e l l i n response. There i s probably not enough, even allowing for 100% absorption through the leaf surface, to a l t e r the physiology of a whole plant i n the f i e l d or greenhouse. 46 Auxins: Like g i b b e r e l l i n s , auxins appear to be produced by a wide range of micro- and macroscopic algae. Mowat (1964) -1 described auxin-like a c t i v i t y of about 1-20 ug kg fresh weight from seven diverse a l g a l sources, including a sample of Maxicrop. Previously van Overbeek (1940) had shown 0.05--1 . .5 ug kg auxin-like a c t i v i t y i n several Phaeophyta, including Macrocystis p y r i f e r a , Desmerestia, Fucus, A l a r i a , Hedophyllum and Laminaria, as well as i n some diatoms and Rhodophytes. As was the case for g i b b e r e l l i n s , auxins appear to be too l a b i l e to survive the rigors of manufacture and storage to which most seaweed products are subjected. Auxins and t h e i r indole derivatives are notoriously photosensitive, and are also l o s t through chemical and enzymatic decomposition (Mann and Jaworski, 1970; Goodwin, 1978). Williams et a l . (1981) checked three commercial seaweed preparations ( A l g i f e r t , Maxicrop and SM-3) and found no trace of auxin a c t i v i t y even i n freshly-prepared products. This observation was contradicted by Sanderson and Jameson (1986) who used gas chromatography-mass spectroscopy to i d e n t i f y unequivocally indole acetic acid (IAA) i n Maxicrop solution freshly prepared from dehydrated powder. Kingman (1975) obtained p o s i t i v e auxin bioassay responses from Ascophvllum nodosum dehydrate used as f o l i a r spray. In an extension of t h i s work, Kingman and Moore (1982) detected IAA i n dehydrated Ascophyllum nodosum powder using gas chromatographic techniques , and estimated the IAA content at -1 -1 50 mg g dehydrate, or approximately 5g IAA kg fresh weight of seaweed. This value i s c l e a r l y orders of magnitude i n excess of the IAA content reported for any other t i s s u e . Bean leaf, for comparison, contained about 0.008 mg kg , while maize endosperm, p a r t i c u l a r l y r i c h i n IAA, contained -1 100 mg kg (Krishnamoorthy, 1982). Thus, the results reported by Kingman and Moore (1982) must be regarded with caution. Cytokinins: Of the main classes of phytohormones to which the growth-promoting a c t i v i t y of seaweed f o l i a r sprays can be attributed, cytokinins seem to be the most l i k e l y candidates. The presence of c y t o k i n i n - l i k e compounds i n macro-algal tissues was determined by Husain and Boney (1969) who bioassayed extracts of Laminaria. Pederson (1973) demonstrated the ef f e c t of exogenous cytokinins on growth of seaweeds i n culture, thus supporting the suggestion that cytokinins may play a regulatory role i n algae similar to that i n higher plants. Cytokinin-like a c t i v i t y has been demonstrated i n extracts of Enteromorpha (Augier, 1972), Ecklonia, Hypnea (Jennings, 1969) , and -Fucus, (Kentzer et. a l . , 1980), as well as many other macrophytic and microphytic species (Augier, 1976). 48 Van Staden's laboratory has issued a unique series of papers i n which the c y t o k i n i n - l i k e components of Sarcrassum  heterophyllum and Ecklonia maxima were i d e n t i f i e d and quantified over time. The l a t t e r seaweed i s used i n the manufacture of the commercial seaweed extract Kelpak. Levels of endogenous cytokinins i n Saraassum were measured and peak levels were found to correlate with gamete i n i t i a t i o n and release. These changes were also associated with t i d a l rhythms and thus had a lunar p e r i o d i c i t y . Higher cytokinin levels were found i n reproductive than i n to vegetative tissue, and o v e r a l l increases i n cytokinin content occurred during the f i r s t quarter lunar phase ( i . e . 24-48 hours after the new and f u l l moons). Maximum cy t o k i n i n - l i k e -1 a c t i v i t y was about 25 ug kg fresh weight (Mooney and van Staden, 1984a). Seasonal changes in the c y t o k i n i n - l i k e a c t i v i t y of t h i s phaeophyte were also evident (Mooney and van Staden, 1984b). The authors reported changes i n the spectrum of c y t o k i n i n - l i k e materials present, as well as i n t h e i r concentrations over the course of the year. Holdfast material contained high levels of material corresponding to the g l y c o s i d i c cytokinins i n winter, whereas during the spring stipe and frond c y t o k i n i n - l i k e a c t i v i t y increased u n t i l receptacle i n i t i a t i o n , then decreased during receptacle growth and development. In a subsequent paper (Mooney and van Staden, 1987a) high performance l i q u i d chromatography (HPLC) was used to t e n t a t i v e l y i d e n t i f y isopentenyl adenine (44% of t o t a l ) , zeatin (11.4%), r i b o s y l z e a t i n (16.9%), and dihydrozeatin (19%) as the cytokinins present. Seasonal v a r i a t i o n i n the l e v e l of endogenous cytokinin-l i k e a c t i v i t y of Ecklonia maxima (Kelpak) was also investigated (Featonby-Smith and van Staden, 1984) . Qualitative and quantitative changes i n cy t o k i n i n - l i k e a c t i v i t y occurred over the year, with low cy t o k i n i n - l i k e a c t i v i t y during periods of active growth, and a gradual increase i n leve l s during the winter. During the summer, substances with the chromatographic (HPLC) c h a r a c t e r i s t i c s of zeatin and r i b o s y l z e a t i n and t h e i r dihydro derivatives dominated, whereas in winter the cytokinin-glycoside f r a c t i o n contained the bulk of the cy t o k i n i n - l i k e a c t i v i t y . The f i r s t suggestion that cytokinins were the active components in seaweed sprays was made by Booth (1969), who pointed out the s i m i l a r i t i e s between responses to kelp f o l i a r sprays and cytokinin f o l i a r sprays. It was not u n t i l 1973, however, that Brain et a l . (1973) performed bioassays on SM-3, a commercially available seaweed spray. They reported that the carrot (Daucus carota) explant cytokinin bioassay response to a 1.3% solution of SM-3 was about half that of a _ i 1 mg 1 solution of ki n e t i n . Higher concentrations of seaweed f a i l e d to promote carrot c a l l u s growth. Later, Williams et a l . (1981) compared cy t o k i n i n - l i k e 50 a c t i v i t y i n three seaweed products: SM-3, Maxicrop, and Marinure. Their results, based on the radish leaf expansion -1 bioassay, indicated that Marinure contained 10-50 mg 1 - i k i n e t i n equivalents, Maxicrop 25-200 mg 1 , and SM-3 15-250 -1 mg 1 . They noted large differences i n c y t o k i n i n - l i k e a c t i v i t y between batches of the same product. The levels of cytokinin reported by Williams et a l . (1981) seem to be rather high when compared to the c y t o k i n i n - l i k e a c t i v i t y reported both i n algae (see previous discussion above) and i n higher plant tissues. For example, among higher plants the highest reported l e v e l of c y t o k i n i n - l i k e a c t i v i t y i s -1 , represented by 350 ug kg i n Zea mays roots (Bearder, 1980). The radish leaf expansion bioassay used by Williams et a l . , (1981) to obtain these estimates of cytokinin content may give an overestimation i n cases where the media on which the leaf disks are floated i s high i n s a l t s , or of unknown composition (Fox and Erion, 1975). Since seaweeds are normally quite high i n s a l t s , i t may be that the seaweed extracts bioassayed contained enough residual s a l t , or other substance, to exaggerate the bioassay response. More rigorous methods of analysis were used by Sanderson and Jameson (1986), who i d e n t i f i e d zeatin, isopentenyl adenine, and isopentenyl adenosine i n Maxicrop by gas chromatography-mass spectroscopy. They also found indications of r i b o s y l z e a t i n glucoside. The t o t a l cytokinin content of the product was estimated at 5.4 ug g dry -1 weight, or about 1.3 mg 1 : enough, according to the authors, to have physiological a c t i v i t y at the applied rates. The following study was ca r r i e d out to define the response of a representative f i e l d crop, Phaseolus vulgaris L. cv Benton, to f o l i a r application of the coastal B r i t i s h Columbia seaweed Macrocystis i n t e g r i f o l i a Bory, within the context of potential economic development of the seaweed resource. In addition, the phytohormone a c t i v i t y of the seaweed was evaluated to t r y to determine whether or not such compounds could account for the b i o l o g i c a l a c t i v i t y of the seaweed spray. 2. MATERIALS AND METHODS 2.1 KELP COLLECTION Samples of kelp (Macrocystis i n t e g r i f o l i a ) were obtained from a single s i t e at Execution Rock near Bamfield, B.C.. Collections were made by boat at approximately the same time each month from September 1983 to September 1984. Additional material was c o l l e c t e d i n May 1985. At each harvest, the 12 apical blades of 12 (summer) or 24 (winter) plants were hand-harvested, torn into pieces 52 (<10 cm) and well mixed. From t h i s composite, grab samples were taken, placed i n zip-lock bags and stored on ice u n t i l they reached Bamfield Marine Station (ca. 2 hrs). There, samples were frozen overnight at -20°C and transported on ice to Vancouver where they were stored at -70°C u n t i l processing. 2.2 KELP PROCESSING Prior to use, kelp was removed from the freezer and broken into pieces which were then passed through a hand-operated kitchen meat grinder. Ground kelp was then loaded into an EDEBO X PRESS (AB BIOTEC,), the barrel of which was pre-cooled to about -20°C i n an 80% (v/v) aqueous methanol-dry ice bath. The kelp was disintegrated by application of about 14,000 kPa, which forced the frozen plant material through a small aperture. Shear forces, as well as pressure-induced changes in the conformation of i n t r a c e l l u l a r ice c r y s t a l s , reduced the kelp to a slur r y which was composed of p a r t i c l e s and c e l l fragments small enough to spray. In an e f f o r t to process the kelp using methods more amenable to eventual commercial production of large quantities, a second process was developed. In t h i s case, kelp, ground by the meat grinder, was passed through a centrifuge homogenizer , followed by passage through a two-piston milk homogenizer (APV Gaulin Inc., Everett Mass.) at -1 250 kg cm . This process produced a l i q u e f i e d kelp similar to that produced by the X PRESS. Subsequent f i e l d t r i a l s indicated that there was no difference i n e f f i c a c y between kelp slurr y produced by the X PRESS and kelp slurr y produced in the three-stage p a r t i c l e reduction process (Temple et a l . 1986). Kelp s l u r r i e s were returned to the -70°C freezer u n t i l used. 2.3 GREENHOUSE EXPERIMENTS A series of greenhouse experiments was conducted to assess the effects of various seaweed extract treatments on growth and y i e l d of beans (Phaseolus vulgaris cv Benton). In general, the experiments were of similar design. A l l were f a c t o r i a l s , i n which one factor was the nature of the kelp f o l i a r spray and the other was s o i l water p o t e n t i a l . Treatments included s o i l s maintained at s p e c i f i c moisture levels (dry, optimal or f i e l d capacity, and waterlogged) by d a i l y weighing. This factor was included because early experiments indicated that, although the bean plants reacted favorably to seaweed application in the f i e l d , the response of optimally watered greenhouse plants was much reduced (Temple et aJL. 1986) . Thus, in an e f f o r t to duplicate more closely the root stresses imposed on f i e l d grown plants, water stresses were applied. The pa r t i c u l a r s of each experiment are described below. 2.3.1 Date of Kelp Harvest: Since many i f not a l l kelp constituents vary i n quality and quantity over time (Whyte and Englar, 1975), an experiment was conducted to determine the temporal v a r i a t i o n of b i o l o g i c a l a c t i v i t y i n the seaweed. A f a c t o r i a l experiment randomly arranged within f i v e blocks was performed. One factor consisted of f o l i a r sprays made from kelp harvested throughout i t s annual l i f e cycle: A p r i l 16, May 13, July 19, September 9, November 10, and January 14. The second factor was water potential (three l e v e l s ) ; s o i l designated as "dry" was maintained between -120 and -150 kPa, " f i e l d capacity" at -30 to -50 kPa, and "wet" at 0 to -10 kPa by da i l y weighing of each "dry" and " f i e l d capacity" pot and addition of appropriate quantities of tap water. "Wet" pots were kept i n shallow basins of water. Five bean seeds were pre-inoculated with Rhizobium  leguminosarum biovar phaseoli (Nitragin), planted to a depth of 1 cm i n 15 cm diameter by 18 cm deep p l a s t i c pots containing steamed, f e r t i l i z e d (145 mg 0-0-60, 290 mg 11-55-0) potting s o i l , and watered l i g h t l y . Most seeds had germinated by day 5, at which time the pots weighed and 55 brought to f i e l d capacity d a i l y by addition of tap water. On day 11 the plants were thinned to two per pot, and the f i r s t f o l i a r sprays were applied. A l l pots to receive a given treatment were removed from the bench and grouped together at some distance from the remaining plants i n order to avoid cross-contamination. Plants were then sprayed with a 1:250 (w/v) d i l u t i o n of seaweed slurry in tap water. Hand sprayers were used, and sprays were applied u n t i l run-off from the leaves occurred. Sprayed plants were then returned to t h e i r bench positions. On day 16 the s o i l water potential treatments described above were imposed, and on day 18 the second seaweed application was made. The bean plants were also staked at t h i s time. The t h i r d and f i n a l seaweed application occurred on day 29, and a l l plants were returned to f i e l d capacity from day 31 to harvest (day 50). Fresh weights of leaves, pods, stems, and leaf areas (LiCor Li-3100, l e a f area meter) per pot were recorded. Dry weights of these y i e l d components, as well as those of the roots following careful extraction and washing i n water were also recorded subsequent to drying to a constant weight at 60°C. Y i e l d variables calculated from these primary data included shoot/root r a t i o , s p e c i f i c leaf area, leaf area r a t i o , t o t a l plant weight, and lea f and stem weight. These growth parameters were subjected to analysis of 56 variance and comparison of treatment means with single degree of freedom orthogona,l contrasts. Trend analysis for date of harvest was p a r t i t i o n e d into l i n e a r , quadratic, cubic and residual components. 2.3.2 Shelf L i f e : The compound(s) i n seaweed sprays responsible for t h e i r growth effects appear to be quite stable, as evidenced by t h e i r persistence throughout rigorous manufacturing, storage, and handling procedures. To determine the longevity of the active components i n a f o l i a r spray derived from M. i n t e q r i a o l i a , an experiment was ca r r i e d out to evaluate the effects of long term storage. Seaweed (M. intecrrigolia) c o l l e c t e d i n May 1984 was processed as described above, and amended with 10% water (v/v), 0.3% (w/w) ammonium phosphate, and 0.1% (w/w) sodium benzoate to prevent b a c t e r i a l and fungal growth (P. Townsley, Dept. of Food Science, University of B r i t i s h Columbia, personal communication). Four 100 ml aliquots were'removed and put into p l a s t i c bottles, one of which was placed i n a -70°C freezer. The others remained at 20°C. At day 118, 250, and 359, one bottle was removed from storage to the -70°C freezer. The e f f e c t of storage at room temperature for these durations on the e f f i c a c y of the f o l i a r spray was tested on bean plants i n a f a c t o r i a l experiment, using f i v e r e p l i c a t e s , the four storage durations, and the three s o i l water potentials described previously. The treatments were completely randomized. The experiment was car r i e d out for 50 days, beginning in May, 1985. Planting, watering, treatment application, and harvesting schedule, as well as greenhouse conditions were i d e n t i c a l to those described above for the date of kelp harvest experiment. Fresh weights of leaves, beans, stems, and leaf areas (LiCor Li-3100, leaf area meter) per pot were recorded. Dry weights of these y i e l d components, as well as those of the roots following careful extraction and washing in water were also recorded subsequent to drying to a constant weight at 60°C. Y i e l d variables calculated from t h i s primary data included shoot/root r a t i o , s p e c i f i c l e a f area (SLA), leaf area r a t i o (LAR), t o t a l plant weight, and leaf and stem weight. Data were analyzed by analysis of variance, with trend analysis performed on length of storage at room temperature against the variables l i s t e d above. 2.3.3 Fractionation of Kelp Extract: A t h i r d greenhouse experiment was conducted to determine whether the growth-promoting effects of the kelp f o l i a r spray could be attributed to compounds within a s p e c i f i c f r a c t i o n obtained by solvent p a r t i t i o n of a methanolic kelp extract. The method of Rademacher and Graebe (1984) was used to 58 obtain f i v e fractions of kelp c o l l e c t e d i n May, 1984, each of which contained a spectrum of compounds with similar s o l u b i l i t i e s i n the p a r t i t i o n solvents. The fractionation procedure was designed to separate groups of phytohormones, but since no fractions were discarded, the entire complement of compounds present in the kelp was applied to test plants. Details of the p a r t i t i o n i n g scheme are presented i n Section 2.4.1, but the phytohormones and some other classes of compounds anticipated i n each f r a c t i o n were: Fraction 1: most a c i d i c phytohormones and other organic acids, auxin, g i b b e r e l l i n s Fraction 2: neutral l i p i d s and pigments, as well as some l i p o p h y l l i c phytohormones such as methylthioisopentenyl adenine and methyl esters of indole acetic acid (IAA) Fraction 3: inorganic s a l t s , sugars, polar g i b b e r e l l i n s , glucosides of a c i d i c phytohormones, some cytokinin ribotides Fraction 4: cytokinins and cytokinin ribosides, g i b b e r e l l i n glycosyl esters, nucleic acid bases, sugars and inorganic s a l t s Fraction 5: residue remaining following methanolic extraction of tissue combined with the a c i d i c aqueous phase of an a c i d i c ethyl acetate p a r t i t i o n . Additional treatments included seaweed spray made from seaweed co l l e c t e d i n May, di l u t e d 1:250 (w/v) with tap water, and a tap water control. The experiment was designed as a f a c t o r i a l using randomized blocks with seven replicates and two factors. One factor was s o i l water potential as described for previous experiments. The second factor was f o l i a r application of the control and seaweed preparations l i s t e d above, at concentrations equivalent to that of the complete or unfractionated seaweed spray. On June 18, 1986, p l a s t i c pots (15 cm diameter, 18 cm deep) were planted with f i v e bean seeds inoculated with Rhizobium leauminosarum biovar phaseoli (Nitragin). These were l i g h t l y watered d a i l y u n t i l day 9, when emerged seedlings were thinned to two uniform plants per pot. On day 16 the water stresses were applied as described for previous experiments, and the f i r s t spray treatments applied to run-o f f . From t h i s time, pots were weighed and brought to t h e i r prescribed water pote n t i a l on a da i l y basis. The second spray application occurred on day 30, and plants were staked up on day 32. The t h i r d and f i n a l f o l i a r treatment was applied on day 44. Relative humidity i n the greenhouse varied between 65% and 85%, with a mean of about 75%. The temperature ranged between 20°C and 36°C, and averaged about 25°C. 60 A l l plants were harvested on day 56. Measured variables included leaf area (Licor Li-3100 leaf area meter), leaf, pod, stem and root fresh and dry weights, number of pods, and stem length and diameter (midpoint, f i r s t internode). Calculated variables were root/shoot r a t i o , s p e c i f i c l e a f area, and leaf area r a t i o . Data were analyzed by analysis of variance using i n d i v i d u a l degree of freedom orthogonal contrasts. 2.4 PHYTOHORMONE ANALYSIS As described in the Introduction and survey of the l i t e r a t u r e (Section 1), i t was apparent that a potential explanation of the eff e c t of seaweed f o l i a r application on plants could be the presence of endogenous phytohormones i n the seaweed extracts. Extraction, p u r i f i c a t i o n , i s o l a t i o n , bioassay, and i d e n t i f i c a t i o n of the phytohormones i n samples of kelp were attempted. Although i n commercial extracts i t i s doubtful that the more l a b i l e phytohormones are present i n s u f f i c i e n t quantity, they were assayed in t h i s study because kelp was stored u n t i l use under conditions through which such compounds could p e r s i s t . 61 2.4.1 Bioassays: In order to provide a way of screening plant extracts for b i o l o g i c a l a c t i v i t y , bioassays for gib b e r e l l i n s , auxins and cytokinins were established. Gi b b e r e l l i n Bioassay: The g i b b e r e l l i n bioassay of Murakami (1968) with s l i g h t modification, was performed as follows. Seeds of r i c e (Oryza sativa cv. Tan Ginbozu) were surface s t e r i l i z e d i n 20% (v/v) commercial bleach for 20 minutes, then rinsed well several times i n s t e r i l e d i s t i l l e d water. Seeds were germinated i n aerated, s t e r i l e d i s t i l l e d water at 30°C for 72 hours. Uniformly sprouted seeds were placed on 1% (w/v) water agar i n 100 X 80 mm glass P e t r i dishes. Each dish was divided into quadrants, and six seeds were planted per quadrant. Seedlings were then grown i n the closed dishes for 48 hours at 30°C under constant illumination. P r i o r to application of a "microdrop" (1 ul) of test solution or GA^ to the base of each co l e o p t i l e , the two least uniform seedlings were removed from each quadrant. Microdrops were dispensed from a 5 u l Unimetrics glass syringe. A l l four remaining plants i n each of three randomly selected quadrants were treated with each solution. Following application of test solutions or standards, seedlings were placed i n constant illumination for 72 hours, after which the length of the second leaf sheath was measured to the nearest millimeter. 62 The equation for the standard curve was established by line a r regression. Test solutions e l i c i t i n g growth responses in excess of the control ( d i s t i l l e d water) were i d e n t i f i e d by cal c u l a t i n g Fisher's protected LSD, and an estimate of the g i b b e r e l l i n a c t i v i t y in each test solution was obtained by substituting the sheath length for those treatments into the standard regression equation using the methods of Zar (1984). Auxin Bioassay: The auxin-like a c t i v i t y i n kelp extracts was determined by the methods of Nitsch and Nitsch (1956). S t e r i l e (20% (v/v) commercial bleach, 60 min.), well-rinsed seeds of Avena sativa cv Victory were germinated i n moist Vermiculite for 72-96 hours i n the dark, at room temperature. A l l further procedures were car r i e d out under dim red l i g h t (CBS Red S a f e l i g h t ) . The apical 3 mm of each hypocotyl were removed with a razor blade, and the subapical 6 mm section was excised and floated on 10 mM phosphate buffer (pH 4.5) containing 2% (w/v) sucrose before use. Test solutions were dried and taken up i n 6 ml of 10 mM phosphate buffer (pH 6.4, 2% sucrose (w/v)), which was equally subdivided into 3 P e t r i dishes (28x10 mm). Similar volumes of buffer containing standard IAA concentrations were also prepared and dispensed. Five hypocotyl sections were floated on the solution in each P e t r i dish. Dishes were incubated i n the dark at room temperature on a rotary shaker 63 to prevent c u r l i n g . After 20 hours, the length of each hypocotyl section was measured with an o p t i c a l micrometer. When the hypocotyl length exceeded that of the control by a si g n i f i c a n t amount as assessed by the LSD, the length was substituted into the regression equation for the standards to obtain an estimate of the auxin-like a c t i v i t y of the extracts. Cytokinin Bioassay: Two cytokinin bioassays were used. I n i t i a l experiments u t i l i z e d the Amaranthus bioassay, which r e l i e s on the measurement of cytokinin-induced betacyanin production (Biddington and Thomas, 1973). Some extracts, were evaluated using t h e i r a b i l i t y to induce c a l l u s growth i n soybean, since t h i s bioassay was better established, easier to perform, and constitutes one of the tests by which cytokinins are defined (Miller, 1965). Amaranthus Betacyanin Bioassay: Amaranthus caudatus seeds were germinated at 25°C in the dark on Whatman 3MM f i l t e r paper for 72 hours. Seed coats were removed from germinated seeds, and ten seedlings were placed i n each 35x10mm P e t r i dish. P e t r i dishes contained Whatman #3 f i l t e r paper discs moistened with 2 ml of the test solutions or standards. Extracts to be tested were dried and taken up i n 13 mM phosphate buffer (pH 6.3) containing 1 mg/ml tyrosine. Seedlings were incubated i n the P e t r i dishes for 18 64 hours at 25°C i n the dark. Subsequently, the seedlings i n each dish were placed i n 3 ml d i s t i l l e d water i n a polypropylene 13x100mm test tube and frozen, then thawed. This freeze-thaw cycle was repeated, and the tubes were agitated before the absorbance of the re s u l t i n g solution was measured at 542 nm and 620 nm using a Perkin Elmer model 552 spectrophotometer. The r e l a t i v e quantity of betacyanin extracted by the cycles of freezing and thawing was quantified by ca l c u l a t i n g the difference i n absorbance at these wavelengths. This quantity was l i n e a r l y related to the cytokinin content of the standard solutions (Biddington and Thomas, 1973). Cytokinin-like a c t i v i t y greater than the control (Fisher's protected LSD) in test extracts was estimated using a lin e a r regression of concurrently assayed standards. Soybean Callus Bioassay: The bioassay of M i l l e r (1960) was used to determine c y t o k i n i n - l i k e a c t i v i t y . Soybean (Glycine  max cv Acme) seeds were surface s t e r i l i z e d i n 10% (v/v) commercial bleach for ten minutes, then well rinsed i n s t e r i l e d i s t i l l e d water. S t e r i l i z e d seeds were then placed on phytohormone-free culture medium i n 500 ml flasks, which were kept at 28°C under constant fluorescent l i g h t . After about two weeks growth, the cotyledons of healthy seedlings were removed a s e p t i c a l l y . Blocks of tissue were excised from the cotyledons and placed on growth media containing 2.5 mg 65 1 kinetin, and 2 mg 1 NAA. The re s u l t i n g c a l l u s was maintained i n the dark at 30°C and sub-cultured to provide bioassay material. Column eluates to be bioassayed were divided i n half, and each half was placed i n a 125 ml Erlenmyer flask. These samples were then dried overnight i n a forced a i r oven at 35°C. To assay Rf zones of paper chromatographs, r e p l i c a t e sections of each zone were a i r dried, then placed into each flask. Twenty ml of cytokinin-free growth medium containing -1 2 mg 1 NAA were added to each flask, which was then stoppered with cotton and autoclaved (20 min., 15 p s i , 220°C). Four pieces of healthy white cal l u s tissue (2mm x 2mm) were then transferred a s e p t i c a l l y to each flask (4 flask S . These were incubated for two weeks at 30°C i n the dark. Total c a l l u s weight per flask was determined, and compared to those from a s e r i a l d i l u t i o n of isopentenyl adenine to allow estimation of cytokinin a c t i v i t y for each test solution. A l i n e a r equation r e l a t i n g response to concentrations of isopentenyl adenine was established and mean responses s i g n i f i c a n t l y d i f f e r e n t from the control (no cytokinin) according to Fisher's protected LSD were substituted into the equation to y i e l d estimates of r e l a t i v e cytokinin a c t i v i t y . 66 2.4.2 Phytohormone Extraction and P u r i f i c a t i o n : Extraction procedures worked out for t e r r e s t r i a l higher plant tissues were tested and adapted to the seaweed. Some protocols resulted i n extracts that were too tarry or sal t y to bioassay or further p u r i f y with confidence. Others were so complex and multi-step that anticipated losses at each step were pro h i b i t i v e , both i n terms of chemical degradation over the time necessary to complete the procedures, and unavoidable losses through p a r t i t i o n i n e f f i c i e n c y and adsorption to glassware at each step. Eventually compromise extraction procedures were adopted, one for auxins and g i b b e r e l l i n s , and three for cytokinins; these were r e l a t i v e l y simple to perform, yet provided fractions that were appropriate for further p u r i f i c a t i o n . A l l solvents were obtained.from BDH, and were of the highest available purity. Methanol, ethyl acetate and n-butanol were g l a s s - d i s t i l l e d immediately p r i o r to use. Aqueous methanol (80%) used for extraction contained 125 mg 1 butylated hydroxy-toluene as an anti-oxidant. A l l glassware was treated with 1% dimethyl dichlorosilane i n benzene (BioRad). Extraction, p a r t i a l p u r i f i c a t i o n and fractionation procedures were developed based on published p a r t i t i o n c o e f f i c i e n t s of the classes of phytohormones under 67 investigation (Hemberg,1974; Hemberg and Westlin, 1973; Horgan, 1978; Yokata et a l . , 1980). This enabled the extraction of auxins, cytokinins and gi b b e r e l l i n s from a single sample, rather than using separate protocols for each. The methods of Rademacher and Graebe (1984) were used to evaluate seaweed samples co l l e c t e d i n May, July and September. Seaweed co l l e c t e d i n May was further analysed by the methods of Taylor et. a l . (1982) and Mukherji and Waring (1983) . Analysis of Monthly Samples: The methods of Rademacher and Graebe (1984) were used. Tissue of Macrocystis i n t e g r i f o l i a processed as described above (Section 2.2) was removed from the -70°C freezer, crushed, and added to methanol (l/4:w/v). Tissue was blended i n a stainle s s s t e e l container immersed i n ice water for 3 min. at high speed using a Sorvall Omni-Mixer. The re s u l t i n g sl u r r y was s t i r r e d at 4°C for four hours, vacuum f i l t e r e d through Whatman #3 f i l t e r paper, which was then rinsed with a small volume MeOH, and the residue re-extracted i n 1/4 (original wt./v) 80% (v/v) aqueous MeOH at 4°C for four hours. The f i l t r a t i o n procedure was repeated and the residue s t i r r e d with a similar volume of 80% MeOH overnight at 4°C. The three f i l t r a t e s were then combined, • and reduced to the aqueous phase i n a Buchi rotavapor. The extract was taken to pH 2.5 with 1 N HCI, and 68 p a r t i t i o n e d four times against equal volumes of water-saturated ethyl acetate. The a c i d i c ethyl acetate phase was reduced i n volume, then p a r t i t i o n e d four times against 1/4 volumes of 0.01 M I^HPO^. This aqueous phase was rotary evaporated (Buchi rotavapor) b r i e f l y to remove residual ethyl acetate, then s l u r r i e d with 15% (w/v) insoluble polyvinylpyrrolidone (PVP) (Sigma, St. Louis) p r e - p u r i f i e d by the method of Harrison (1973). PVP was removed by vacuum f i l t r a t i o n , and washed with a small volume of I^HPO^, which was added to the f i l t r a t e . The PVP step was repeated once or twice, u n t i l the f i l t r a t e was clear. The c l a r i f i e d f i l t r a t e was adjusted to pH 3.0 with IN HCl, and part i t i o n e d four times with 1/2 volumes of ethyl acetate. This a c i d i c ethyl acetate phase, expected to contain auxins and gi b b e r e l l i n s , was reduced i n volume and taken up i n acetone or methanol p r i o r to chromatography. Residual ethyl acetate was removed from the o r i g i n a l aqueous phase (Buchi rotavapor) , and adjusted to pH 7.8 with IN KOH p r i o r to p a r t i t i o n i n g f i v e times with 1/5 volumes of water- saturated n-butanol. The butanolic phase, expected to contain the cytokinins and t h e i r ribosides, was taken to dryness (Buchi rotavapor at less than 35°C), and taken up i n 50 ml of 50% (v/v) ethanol p r i o r to chromatography on Dowex 50X (Sigma, St. Louis) according to Van Staden (1976). A summary of t h i s proceedure i s presented i n Figure 1. This macerate tissue extract i n 80% MeOH (x3) vacuum f i l t e r combine f i l t r a t e s reduce to aqueous pH 2.5 (IN HCI) p a r t i t i o n (x4) with equal v o l . EtOAc EtOAc p a r t i t i o n (x3) with 1/4 v o l . 0.01M K 2 H P O 4 residue aqueous I PVP s l u r r y pH^.O (IN HCL) p a r t i t i o n (x4) with 1/2 v o l . EtOAc EtOAc aqueous EtOAc Fraction I Fraction II aqueous pH 7.8 (IN KOH) p a r t i t i o n (x5) with 1/5 v o l . n-butanol I aqueous n-butanol Fraction III Fraction IV Fraction V FIGURE 1 Flow diagram of the extraction methods of Rademacher and Graebe (1984). Fraction I would contain auxin and g i b b e r e l l i n . Fraction IV most cytokinins. 70 yielded f i v e fractions which were i d e n t i c a l to those used i n the kelp fractionation greenhouse experiment (Section 2.3.3). To t r y to estimate losses during extraction and chromatography, 1.0 mg each of commercially obtained IAA (indoleacetic acid), isopentenyl adenine, and isopentenyl adenosine in 1 ml water were added to 80% (v/v) aqueous methanol and extracted as though i t were a seaweed sample. The IAA-containing fra c t i o n , (Fraction I, Figure 1) was dried by rotary evaporation (Buchi rotavapor at less than 35°C), and taken up in 1 ml methanol, the same volume as o r i g i n a l l y processed. F i f t y u l were then taken and analyzed by g a s - l i q u i d chromatography as described i n Section 2.4.3. Peak areas corresponding to the standards were then compared to those from a 50 u l sample of the o r i g i n a l stock solution. To estimate losses of IAA during th i n layer chromatography (TLC), 500 ug of standard IAA i n 500 u l of methanol were applied to TLC plates and run i n isopropanol/ammonia/water (10/1/1:v/v/v). Rf zones from 0.1 to 1.0 were scraped o f f and eluted as per Section 2.4.3. The eluates were dried under a stream of nitrogen, and the residues taken up in 0.5 ml acetone. A 50 u l aliquot was removed to a R e a c t i v i a l and analyzed by GLC. Again peak areas corresponding to that of IAA were compared to that of a sample of the o r i g i n a l stock solution. 71 S i m i l a r l y the n-butanolic phase (Fraction IV, Figure 1) was dried, taken up in the o r i g i n a l sample volume, and compared by GLC to an equal volume of the o r i g i n a l stock solution. Further Analysis of Samples Collected i n May: To t r y to i d e n t i f y t e n t a t i v e l y any phytohormonally active substances i n 50 gm of seaweed co l l e c t e d i n May, two cytokinin-containing fractions of seaweed extracts obtained by the methods of Mukherji and Waring (1983) (Figure 2) were chromatographed on an XAD-2 column as described i n Section 2.4.3. Elution c h a r a c t e r i s t i c s of standard cytokinins were established by running these compounds, drying the fractions described i n Section 2.4.3, and analyzing the residues by GLC as described in Section 2.4.3. Fifte e n ml fractions of each of the two seaweed extracts were co l l e c t e d and pooled to y i e l d six fractions which were rotary evaporated (Buchi rotavapor at less than 35°C) to near dryness, and taken up i n 80% aqueous MeOH (v/v). Half of each of these was bioassayed i n the Amaranthus cytokinin bioassay (Section 2.4.1), while the remainder was dried under a stream of nitrogen and analyzed by GLC as described i n Section 2.4.3. One u l of the b i s - t r i m e t h y l s i l y l trifluoracetamide derivatives from each of the six eluate fractions was injected into the column (Kingman and Moore, 72 ma cerate tissue extract i n 80% MeOH (x3) vacuum f i l t e r combine f i l t r a t e s reduce i n volume (< 35°C) freeze and thaw centrifuge (5,000 xg, 1 hr) supernatant to pH 2.5 (IN HCl) p a r t i t i o n (x4) with equal v o l . EtOAc EtOAc reduce i n volume (< 35°C) I take up i n 80% aqueous MeOH I PVP reduce i n volume (<35°C) take up i n solvent for chromatography aqueous PVP slu r r y I f i l t e r , repeat f i l t r a t e pH 7 (IN KOH) p a r t i t i o n (x4) with 1/2 vol . n-butanol a c i d i c ethyl acetate f r a c t i o n I n-butanol reduce i n volume (<35°C) take up i n s o l v e n t for chromatography n-butanolic f r a c t i o n I aqueous I discard FIGURE 2 Flow diagram of the extraction methods of Mukherji and Waring (1983) . 73 1982), and the r e s u l t i n g chromatograms were examined for peaks with retention times si m i l a r to those of the available standards. Fractions containing such peaks were^spiked' with the appropriate standard and re-chromatographed. Co-elution was defined as enlargement of the peak of interest when co-injected with the standard. Fractions showing co-elution with a standard cytokinin were re-tested using a d i f f e r e n t temperature program on the gas chromatograph (185°C to 265°C, 5°C min )^ to determine i f co-elution was maintained under d i f f e r e n t chromatographic conditions. Peaks showing co-elution under both temperature programs and that appeared i n bioassay-active XAD-2 fractions appropriate to the co-eluting standard were te n t a t i v e l y i d e n t i f i e d as that standard. The methods of Taylor et a l . (1982) were used to analyze May seaweed further for c y t o k i n i n - l i k e a c t i v i t y (Figure 3). Tissue samples were homogenized and extracted three times i n MeOH as described above. The combined f i l t r a t e s were reduced in volume by rotary evaporation (Buchi rotavapor at less than 35°C), then taken to pH 8.0 with 1 N KOH. This was centrifuged at 10000 xg for 15 min., and the supernatant taken to pH 3.0 with IN acetic acid before chromatography on a c e l l u l o s e phosphate column as described i n Section 2.4.3. The ammoniacal eluate was reduced i n volume by rotary macerate tissue extract (x3) i n 80% MeOH combine f i l t r a t e s reduce to aqueous pH 8.0 (IN KOH) centrifuge decant supernatant pH 3 (IN acetic acid) apply to c e l l u l o s e phosphate column ( N H i + form, 2 cm x 15 cm) wash with 3 column volumes H2O (pH 3 with acetic acid) wash with 3 column volumes H zO elute with 8 column volumes 0.3N NH4OH ammoniacal eluate to pH 8 with IN HCl p a r t i t i o n (x4) with equal v o l . n-butanol reduce n-butanol to dryness take up i n solvent for chromatography Figure 3 Flow diagram of the extraction methods of Taylor et (1982). The butanolic f r a c t i o n would contain the cytokinins. 75 evaporation (Buchi rotavapor at less than 35 C), then taken to pH 8.0 with 1 N HCl. It was then pa r t i t i o n e d 4 times against equal volumes of water-saturated n-butanol. The butanolic phase was taken to near dryness by evaporation (Buchi rotavapor at less than 35°C), and the residue taken up in MeOH for paper chromatography, or 35% (v/v) aqueous ethanol for Sephadex LH-20 chromatography (Section 2.4.3). 2.4.3 Phytohormone Chromatography: Gibb e r e l l i n s : Fractions expected to contain g i b b e r e l l i n - l i k e substances i n acetone were streaked onto Whatman 3 MM chromatography paper and developed i n a descending manner i n isopropanol/ammonia/ water (10/1/1:v/v/v) (Majhukar and Waring, 1983). These were a i r - d r i e d and then divided into ten 0.1 Rf regions. One ml of 50% (v/v) aqueous acetone (Murakami, 1968) containing the g i b b e r e l l i n - l i k e substances was eluted from each s t r i p by hanging them from a reservoir of acetone with the lower end of the s t r i p discharging eluate' into a test tube. These were bioassayed as described above (Section 2.4.1). Mobility of a standard, GA^ (Sigma, St. Louis) was determined by v i s u a l i z i n g spots of chromatographed standards under u l t r a v i o l e t l i g h t after spraying with sulphuric acid/ethanol (5/95:v/v) and heating at 100°C for 1 hour (Yokota et a l . , 1980). Auxins: Fractions expected to contain auxin-like substances were streaked onto Kieselgel 60-F254 TLC plates (Merck) which were developed i n chloroform/methanol (17/3:v/v) (Rademacher and Graebe, 1984). Plates were l e f t at room temperature and l i g h t l y sprayed with water to prevent the plates from drying completely as the chromatography solvents evaporated. These plates were cut into ten 0.1 Rf regions which were scraped into 3 ml 10 mM phosphate buffer (pH 6.4, 2% sucrose w/v) and vortexed for auxin-like substance recovery. TLC plate matrix was removed by centrifugation and re-washed with another 3 ml buffer, then re-centrifuged. The combined supernatants were reserved for inclusion i n the bioassay. Cytokinins: Several chromatography systems were used to try to pu r i f y and separate the cyt o k i n i n - l i k e compounds i n extraction frac t i o n s . Dowex 50X: Pr e - p u r i f i c a t i o n was carr i e d out on a Dowex 50x8 3 (Sigma) cation exchange column (2 cm diameter, 135 cm ). Samples were taken up i n 50 ml 80% (v/v) aqueous ethanol adjusted to pH 3.0 with acetic acid, and passed through the column. The column was then washed successively with 300 ml H20, 200 ml 80% ethanol, and 1 1 3M NH^OH i n 50% (v/v) ethanol (Van Staden, 197 6). The ammoniacal f r a c t i o n was 77 dried by rotary evaporation (Buchi rotavapor at less than 35°C) and taken up in a minimal volume of methanol, then applied to a Keiselgel 60-F254 TLC plate. After e q u i l i b r a t i o n , the chromatogram was run i n chloroform/methanol (17/3 v/v) (Rademacher and Graebe, 1984). Scrapings of each Rf zone were washed three times with 3 ml of methanol, centrifuged, and the combined supernatants were divided equally into three P e t r i dishes and a i r dried for bioassay. Mobility of standard compounds (Sigma, St. Louis) was determined by v i s u a l i z a t i o n of chromatographed standards under u l t r a v i o l e t l i g h t (Yokota et a_l., 1980) . Cellulose Phosphate: Al t e r n a t i v e l y , extracts were f i r s t p u r i f i e d on columns of ce l l u l o s e phosphate (Taylor et a l . , 1982). Cellulose phosphate was washed with tap water p r i o r to pouring the column. Impurities were removed by passing 500 ml 0.2M HCl, then 500 ml H2<D, then 500 ml 0. 3N ammonium hydroxide through the column. This was followed by 1 1 H20 and 200 ml ammonium acetate buffer ((5.8 ml acetic acid i n 1 1, adjusted to pH 3.1 with ammonium hydroxide) Taylor,Agriculture Canada, Lacombe Alberta, personal communication). Extracts i n aqueous solution were adjusted to pH 3.0 with ammonium hydroxide, and applied to the column (2 cm X 15 cm). The column was then washed successively with three column volumes of H 20 (pH 3.0 with acetic acid), three column volumes of H20, and then eight column volumes of 0.3N 78 ammonium hydroxide. The ammoniacal eluate was adjusted to pH 8.0 with IN HCI, then pa r t i t i o n e d four times against equal volumes of water-saturated n-butanol. The butanolic phase was rotary.evaporated (Buchi rotavapor) and the residue redissolved in methanol p r i o r to strip-loading onto Whatman 3MM chromatography paper. Descending chromatograms were run in isopropanol/ammonium hydroxide/water (10/1/1:v/v/v) (Taylor et a l . 1982). Strips of the chromatograms representing each Rf region were included in bioassay fl a s k s . Sephadex LH 20: Additional chromatography on Sephadex LH-20 was also performed according to Horgan (1978). Cytokinin extracts were applied to a 2 cm x 30 cm column equilibrated with 35% aqueous ethanol (v/v). A flow rate of about 1 ml -1 . mm. was maintained by using a p e r i s t a l t i c pump, and 40 20 ml fractions were co l l e c t e d and a i r dried before bioassay. Elution volumes of authentic standards were established by monitoring eluate absorbance at 254 nm. XAD-2: A single experiment was performed u t i l i z i n g a column of Amberlite XAD-2 (Sigma, St. Louis) (Stafford et a l . , 1984). Fractions suspected of containing cytokinins were taken to dryness by rotary evaporation (Buchi rotavapor at less than 35°C) and taken up i n 100 ml 0.1 M phosphate buffer (pH 8.0) before application to the column (2 cm x 15 cm). The column was then eluted with a step-wise gradient of 0%, 15%, 50%, and 100% methanol/water (v/v) (Stafford et a l . , 79 1984). Fif t e e n ml fractions were co l l e c t e d and pooled as follows: 0% MeOH 90 ml 15% MeOH 90 ml 50% MeOH A 45 ml 50% MeOH B 45 ml 100% MeOH A 45 ml 100% MeOH B 45 ml Each pooled f r a c t i o n was rotary evaporated to dryness and redissolved i n 10 ml 80% aqueous MeOH (v/v) and divided into two 5 ml aliquots. One was dried under a stream of nitrogen for analysis by gas liquid-chromatography (GLC) (Section 2.4.3, i v ) , the other for incl u s i o n in the Amaranthus cytokinin bioassay (Section 2.4.1). Gas Chromatography: When samples were to be analysed further by gas chromatography they were f i r s t rotary evaporated (Buchi rotavapor at less than 35°C) to about 1 ml before transfer to mic r o s i l a t i o n vessels. Once i n mic r o s i l a t i o n vessels, samples were reduced to dryness under a stream of nitrogen. A l l water was removed azeotropically by repeated addition and evaporation of benzene:acetone (1/1:v/v) (Upper et a l . , 1970). Aliquots (100 ul) of b i s - t r i m e t h y l s i l y l 80 trifluoracetamide (BSFTA) (Sigma) were added to each anhydrous sample, and the s i l a t i o n vessels were sealed using t e f l o n septa and kept at 65°C for two hours (Kingman and Moore, 1982). This procedure yields t r i m e t h y l s i l y l derivatives suitable for gas chromatographic analysis. The derivatized fractions were injected into a 3% SE 30 on Gaschrome Q (100-200 mesh) s i l a n i z e d glass column (2 mm x 6') under the following conditions: N 2 flow rate, 29 ml/min; FID temp., 350°C; in j e c t o r temp., 250°C; oven temp., 185°C for 2.5 min., 30°C min - 1 to 235°C, 235°C for 40 min.. For co-elution determinations samples were also run using a 185°C to 2 65°c temperature program. 81 3. RESULTS 3.1 GREENHOUSE EXPERIMENTS 3.1.1 Date of Kelp Harvest: In t h i s experiment plants were sprayed with homogenized seaweed c o l l e c t e d throughout i t s growing season (April, May, July, September, November and January). Data were subjected to analysis of variance, with trend analysis of y i e l d variables by date of kelp harvest. Results are presented i n Table 3. There were no s i g n i f i c a n t main effects, however the s i g n i f i c a n t interaction (FC/D+W*QUADRATIC) for several variables including l e a f area, leaf dry weight, stem dry weight, shoot dry weight, plant dry weight and shoot/root r a t i o indicate that the responses i n s o i l kept at f i e l d capacity d i f f e r e d according to kelp harvest date compared to plants i n s o i l subjected to moisture stress (ie. dry and wet s o i l treatments). Graphs of the s o i l variables for each month of kelp harvest reveal that a peak in response occurred i n f i e l d capacity s o i l s for kelp harvested i n the July to September period, whereas no such peak occurred for plants grown under s o i l moisture stress conditions. This relationship was similar for leaf area, leaf dry weight, stem dry weight, shoot dry weight and t o t a l plant dry weight. For the shoot/root r a t i o , t h i s peak i n 82 TABLE 3: EFFECT OF KELP SPRAY MADE FROM MACROCYSTIS INTEGRIFOLIA COLLECTED AT VARIOUS TIMES OF THE YEAR ON GROWTH OF PHASEOLUS VULGARIS UNDER 3 MOISTURE REGIMES. MONTH OF KELP COLLECTION DRY SOIL F I E L D CAPCITY SOIL WET SOIL SIGNIFICANT . : : • — — : : — EFFECTS AND VARIABLES APRIL MAY JULY SEPT. NOV. JAN. APRIL MAY JULY SEPT. NOV. JAN. APRIL MAY JULY SEPT. NOV. JAN. . INTERACTION L E A F AREA 3514 3518 3382 3467 3555 3415 3186 3152 3943 3638 3342 3085 2692 2376 2514 2551 2444 2507 DATE QUAD (CM 2 ) a a a a a a a a b ab a b a a a a a . a • a FC/DW*QUAD LEAF DW 10.19 10.42 9.82 10.28 10.31 10.38 9.77 10.31 12.19 10.99 10.12 9.00 8.09 7.311 7.56 8.00. 7.45 8.48 FC/DW*QUAD (G) a a a a a a ab a b b ab ab a a a a a a a STEM DW 7.50 7.66 7.33 7.30 7.97 7.26 7.46 6.82 8.49 8.33 7.15 6.34 5.37 4.92 4.96 5.21 4.91 5.23 FC/DW*QUAD (G) a a a a a a ab ab b b ab a a a a a . a a POD DW 10.41 12.21 11.11 10.52 10.20 9.18 9.63 9.42 10.55 11.11 10.17 9.36 6.89 7.04 6.92 6.94 6.06 7.27 (G) a a a a a a a a a a a a a a a a a a SHOOT DW 28.10 30.29 28.26 28.10 28.48 28.82 26.87 26.55 31.23 30.43 27.44 24.71 20.35 19.27 19.44 20.14 18.42 20.97 FC/DW*QUAD (G) a a a a a a a a a a a a a a a a a a ROOT DW 6.14 7.05 4.94 6.01 6.19 5.42 J5.16 6.25 6.53 4.62 5.94 5.18 5.24 5.83 5.91 6.14 5.38 6.11 (G) a a a a a a ! ab b b a ab ab a a a a a a PLANT DW 34.24 37.34 33.20 34.11 34.68 32.24 32.03 32.80 37.77 35.05 33.39 29.89 25.59 25.10 25.35 26.28 23.80 27.08 FC/DW*QUAD (G) a a a a a a a a a a a a a a a a a a SHOOT/ROOT 5.13 5.19 5.86 5.16 4.72 5.24 5.67 4.41 5.39 6.95 5.20 4.S4 4.00 3.52 3.50 3.48 3.49 3.51 FC/DW*QUAD RATIO (DW) a a a a a a ab a a b a a a a a a a a LAR 102.8 95.4 102.6 103.0 102.9 106.7 99.7 . 96.2 106.6 104.9 99.7 108.7 105.1 95.1 102.5 99.1 103.2 92.97 a a a a a a a a a a a a a a a a a a SLA 348.7 338.9 348.4 348.5 353.6 40.4 337.2 309.8 339.6 334.0 326.9 345.2 334.3 329.9 338.5 328.4 332.4 305.4 (M2 G~ 1 ) a a a a a a a a a a a a a a a a a a G r e e n h o u s e grown Phaseolus vulgaris p l a n t s were s p r a y e d t h r e e t i m e s w i t h l i q u i f i e d Macrocystis integrifolia c o l l e c t e d t h r o u g h o u t i t s l i f e c y c l e . Wet t r e a t m e n t s were k e p t w a t e r l o g g e d , f i e l d c a p a c i t y t r e a t m e n t s a t 0 t o -10 kPa, and d r y t r e a t m e n t s a t -120 t o -150 kPa s o i l w a t e r p o t e n t i a l . Numbers f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t w i t h i n e a c h s o i l w a t e r p o t e n t i a l (LSD, p<0.1). 83 response i n f i e l d capacity s o i l s occurred at the September sampling date. This experiment indicates that there i s l i t t l e e f f e c t of kelp harvest date on plant responses under s o i l moisture stress conditions. On s o i l at f i e l d capacity, however, kelp harvested between July and September exerts a greater e f f e c t on plant growth than samples c o l l e c t e d early i n the spring or l a t e r during the winter months. 3.1.2 Shelf L i f e : In t h i s experiment the effect of storing the homogenized kelp at room temperature was evaluated. Results of trend analysis in ANOVA yielded only two si g n i f i c a n t results and are presented i n Table 4. Both were effects on s p e c i f i c l eaf area. The f i r s t indicates that there was a li n e a r relationship between the length of kelp storage and i t s e f f e c t on SLA. This variable decreased l i n e a r l y from T Q to the f i n a l storage time, at 359 days. Although the relationship was linea r , the t o t a l decrease i n a c t i v i t y was only 4%, and therefore probably of l i t t l e b i o l o g i c a l s i g n i f i c a n c e . Secondly, the FC/D+W*QUADRATIC interaction was s i g n i f i c a n t . A l i n e graph of SLA of FC and D+W indicates that in f i e l d capacity s o i l s 84 85 the 118 day storage time caused a greater increase i n SLA than no storage, or storage exceeding 118 days. At the same time, the D+W curve indicates a s l i g h t depression i n t h i s variable midway through the storage period. Thus the combined experiment-wide SLA approached l i n e a r i t y as the depression of SLA in the moisture stressed s o i l s countered the increases seen in the f i e l d capacity s o i l s . This generated the s i g n i f i c a n t l i n e a r main eff e c t described above. Examination of the variables that comprise SLA reveals that, across a l l water potentials, leaf area (LA), which was non-linear in trend analysis, was highest at TQ, and l e v e l l e d o f f for the remaining time in t e r v a l s (118, 250, and 359 days). Si m i l a r l y leaf (LW) weight was highest for plants treated with kelp stored for zero days, but dropped to i t s lowest point for kelp stored for 118 days. Longer storage times resulted i n treated plants with intermediate leaf weights. Thus the c r i t i c a l point i n the curves (118 days) caused t h e i r product (LA/LW = SLA @ 118 days) to r i s e to a le v e l intermediate between results for kelp stored for 0 and 250 days on the SLA curve, generating a straight l i n e with a slope near zero. In any event, the ov e r a l l F-test for the analysis of variance indicated no differences among treatment means. Thus, i t appears that the storage of the homogenized kelp at 86 room temperature for up to one year does not s i g n i f i c a n t l y a l t e r i t s e f f e c t s on plant growth. The LSD test within each water pote n t i a l revealed few s i g n i f i c a n t differences among ef f e c t s of seaweed aged for d i f f e r e n t time periods. In dry s o i l , the root dry weight of unaged-kelp treated plants was s i g n i f i c a n t l y higher than that of 118-day old kelp treated plants. This resulted i n a concommitant s i g n i f i c a n t reduction i n shoot/root r a t i o as well. Some recovery of t h i s effectiveness was evident, however, since shoot/root r a t i o s of plants treated with kelp stored for 359 days were also greater than those of plants treated with 118 day-old kelp. In f i e l d capacity s o i l , l eaf dry weight was reduced by application of 118 day-old kelp compared to the e f f e c t of applying 359 day-old kelp. As a consequence, SLA of the plants treated with 118 day-old kelp was higher. F i n a l l y , i n wet s o i l , the analysis revealed only one difference among treatment means: height of plants subjected to kelp stored for zero days was s i g n i f i c a n t l y greater than those of other treatments. Thus, two points emerge from t h i s experiment. F i r s t l y , there seems to be a point i n the s h e l f - l i f e of the kelp spray at which i t s effects decrease (118 days) when applied to plants i n dry or f i e l d capacity s o i l s . In dry s o i l s , root dry weight was affected, i n f i e l d capacity s o i l s , l eaf dry 87 weight was a f f e c t e d . In wet s o i l s , p l a n t height decreased with i n c r e a s i n g kelp storage d u r a t i o n . Since the l o s s of e f f e c t i v e n e s s was temporary i n both dry and wet s o i l s , i t may have been caused by a t r a n s i e n t breakdown or intermediate o x i d a t i o n product. Furthermore, the height e f f e c t i n wet s o i l s , which d i d not disappear w i t h f u r t h e r aging of the kelp, i n d i c a t e s the breakdown of some kelp component that s t i m u l a t e d stem elon g a t i o n . 3.1.3 Kelp F r a c t i o n a t i o n : In t h i s experiment the kelp was f r a c t i o n a t e d i n t o f i v e p a r t s by means of solvent p a r t i t i o n i n g , as described i n the M a t e r i a l s and Methods Section (Figure 1). F r a c t i o n s were then a p p l i e d to p l a n t s and i n d i v i d u a l degree of freedom c o n t r a s t s were performed on grouped means. The r a t i o n a l e f o r the grouping of means i s as f o l l o w s . The f i r s t c o n trast compares the e f f e c t s of treatments 3+5 with those of treatments 1+2+4+6+7. This i s a contrast between those f r a c t i o n s which were expected to contain i n h i b i t o r y or non-stimulatory components, wi t h the c o n t r o l plus those f r a c t i o n s expected to contain c y t o k i n i n s , auxins, or g i b b e r e l l i n s , as w e l l as the complete un-extracted kelp homogenate. The next c o n t r a s t , 3 vs 5, compares the e f f e c t of the p u t a t i v e i n h i b i t o r y f r a c t i o n ( F r a c t i o n 3) with that of the c e l l u l a r d e bris remaining a f t e r solvent e x t r a c t i o n of the 88 kelp (Fraction 5). The next contrast, 7 vs 1+2+4+6, compares the e f f e c t of the control with those of the fractions expected to contain stimulatory phytohormones. This group was then subdivided to provide a contrast between the fractions expected to contain auxins (Fractions 1+2) with that expected to contain the majority of the cytokinins (Fraction 4). The f i n a l contrast compares the fr a c t i o n expected to contain free auxin and g i b b e r e l l i n (Fraction 1) with that expected to contain some auxin derivatives (Fraction 2). The selection of these contrasts results i n a set of comparisons which are meaningful i n that differences among putative i n h i b i t o r y and non-inhibitory fractions can be made, followed by further comparisons between the control and putative stimulatory f r a c t i o n s . Comparisons are then made between and within classes of phytohormone-containing fractio n s . Results are presented i n Table '5. Several main ef f e c t s were s i g n i f i c a n t i n t h i s experiment. The comparison between Fractions 3+5 vs 1+2+4+6+7 (putative i n h i b i t o r y fractions vs control+putative stimulatory fractions) yielded s i g n i f i c a n t differences for several variables. These included leaf area, leaf fresh weight, stem fresh weight, number of beans, LAR, and plant height. Leaf area was largely unaffected by Fractions 3+5, with the largest contribution to the difference between these 89 I TABLE 5 : EFFECT FOLIAR SPRAYS MADE FROM EXTRACTS BEAN GROWTH UNDER THREE SOIL MOISTURE REGIMES O F A f . INTEGRIFOLI A O N . TREATMENTS i DRY FIELD CAPACITY WET SIGNIFICANT EFFECTS AND VARIABLES 1 2 3 4 5 6 7 1 2 3 4 5 i 6 7 1 2 3 4 5 . 6 7 LEAF AREA 2963 2932 2901 2859 2981 2974 2976 4030 4394* 3458 3978 3834 3934 3801 3748 3506 3502 4344* 3416 3475 3566 35/12467 (CM* ) (0) (-1) (-3) (-4) (0) (0) (+6) (+16) (-9) ( + 5) (+1) (+3) (+5) (-2) (-2) (+22) (-4) (-3) D/W*4/12 LEAF FW 51.10 53.10 52.15 52.36 52.36 52.87 53.20 " 71.13 75.87* 60.90 70.79 69.10 70.42 67.73 70.65 65.66 61.21 79.65 63.62 63.07 64.16 35/12467 (G) (-4) (0) (-2) (-2) (-2) (-1) (+5) (+12) (-10) ( + 5) (+2) ( + 4) (+1) (+2) (-5) (+24) (-1) (-2) STEM FW 29.21 27.64 26.30 26.14 28.10 28.64 28.26 37.17 41.00 35.49 36.38 34.18* 38.72 (G) (+3) (-2) (-7) (-8) (-1) (+1) (-5) (+4) (-10) (-7) (-15) (-2) 39.32 35.81 34.88 34.87 ( + 6) (+4) ( + 4) 41.30* 35.22 35.22 (+23) (+5) (+5) 33.66 35/12467 D/W*4/12 POD FW 95.17 90.11* 93.31* 90.94* 101.60 102.70 105.30 133.0 149.8 128.9* 135.4 131.1 141.5 142.6' 145.3 139.7 139.9 173.3 150.0 142.4 149.7 D/W*4/12 (G) (-10) (-14) (-11) (-14) (-4) (-2) (-7) (+5) (-10) ' (-5) (-8) (-1) (-3) (-7) (-7) (+16) (0) (-5) SHOOT FW 175.5 170.8 171.8 169.4* 183.0 184.2 186.8 241.3 266.7 225.3* 242.6 234.4 250.6 249.6 251.8 240.2 235.9 294.2* 248.9 240.7 247.5 D/W*4/12 (G) (-6) (-9) (-8) (-9) (-2) (-1) (-3) (+7) (-10) (-3) (-6) (.0) (+2) (-3) (-5) (+19) (+1) (-3) ] LEAF DW 7.62 7.67 7.94 7.79 7.76 7.45 7.40 10.85* 10.80* 9.09 10.39 10.20 10.46 9.41 11.08 9.90 9.30 11.53* 9.57 9.42 9.70 j 7/1246 (G) (+3) (+4) (+7) (+5) (+5) (+1) (+15) (+15 ) (-3) (+10) ( + 8) (+11) (+14) (+2) (-4) (+19) (-1) (-3) i STEM DW 5.73 5.46 5.07 4.99 5.56 5.49 5.42 7.37 8.05 6.76 7.03 6.62 7.98 7.62 6.61 6.51 6.95 7.95* 6.96 6.79 6.43 FC/DW*35/12467 (G) (+6) (+1) (-6) (-8) (+3) (+1) (+3) (+6) (-11) (-8) (-13) (+5) (+3) (+1) (+8) (+24) ( + 8) (+6) FC/DW*4/12 POD DW 15.15 14.58* 13.76* 13.83* 14.25* 15.44* 16.91 18.93 21.95* 18.63 19.76 19.57 20.78 19.07 18.21 16.64 17.54 21.14* 18.70 18.18 16.28 D/W*4/12 (G) (-10) (-14) (-19) (-18) (-15) (-9) (-1) (+15) (-2) (+4) (+3) " -(+.?) (+12) (+2) ( + 8) (+30) (+15) (+12) D/W*7/1246 ROOT DW 4.54 4.85 4.79 4.24 4.52 5.12 4.76 6.14 5.74 5.36 5.87 5.50 5.99 5.39 6.14 6.54 5.91 7.96* 6.19 6.98 5.67 D/W*4/12 (G) (-5) ( + 2) (+1) (-11) (-5) ( + 8) (+14) (+6) (-1) ( + 9) (+2) (+11) ( + 8) (+15) (+4) (+40) (+9) (+23) 7/1246,D/W*4/12 SHOOT DW 46.15 45.58* 44.76* 44.83* 45.25* 46.44 47.91 49.93 52.95* 49.63 50.76 50.57- 51.78 50.07 (G) (-4) (-5) (-7) (-6) (-6) (-3) (0) (+6) (-1) (+1) (+1) (+3) 49.21 47.64 .48.54 52.14* 49.70 49.18 (+4) . (+1) (+3) (+10) (+5) (+4) 47.28 D/W*7/1246 FC/DW*4/12,D/W*l/2 PLANT DW 50.69 50.43* 49.54* 49.07* 49.78* 51.56 52.67 J56.06 58.69* 54.99 56.64 56.08 57.76* 55.45 55.35 54.18 54.45 60.10* 55.88 56.16 52.95 . D/W*7/1246 (G) (-4) (-4) (-6) (-7) (-5) (-2) 1 (+1) (+6) (-1) (+2) (+1) (+4) (+5) (+2) (+3) (+14) (+6) (+6) D/WM/12 SHOOT/ROOT RATIO (DW) 10.78 (+2) 9.77 (-7) 10.17 (-4) 11.93 (+13) 10.25 (-3). 9.54 (-10) 10 .55 j 8.57 (-9) 9.44 (0) 9.49 (+1) 9.72 (+3) 9.85 (+4) 8.82 (-7) 9.44 8.16 : (-6) 7.34 (-15) 8.79-(+2) 6.92 (-20) 8.26 (-5) 7.55 (-13) 8^65 D/W*4/12 # OF PODS 27.8 (+4) 28.8 (+17) 26.0 (-3) 25.2 (-6) 28.0 (+4) 29.4 (+10) 26 .8 39.8* (+25) 44.4* (+40) 28.4 (-11) 33.8 (+6) 3.2.8 (+3) 34.4 ( + 8) 31.8' ! 26.8 I (-4) 27.4 (-2) 26.0 (-7) 35.0 . (+25) 28.8 ( + 3) 28.6 (+2) 28.0 35/12467,7/1246,FC/DW*l/2 ; FC/DW*35/12467,FC/DW*4/12 HARVEST INDEX 0.300 (-6) 0.288* (-10) 0.274* (-14) 0.282* (-12) 0.288* (-10) 0.300 (-6) 0. 320 0.334 (-2) 0.372* (+9) 0.338 (-1) 0.348 (+2) 0.350 (+2) 0.362 (+6) 0.342 i 0.314 (+3) 0.302 (-1) 0.310 (+2) 0.344 (+13) 0.326 (+7) 0.322 ( + 6) 0.304 D/W*7/1246 • D/W*4/12,FC/DW*l/2 LAR 58.43 58.07 58.74 58.32 59.97 57.59 56.47 71.83 75.14 63.03 70.15 68.61 68.23 68.55 ( + 3) (+3) ( + 4) (+3) (+6) ( + 2) (+5) (+10) (-8) ( + 2) (0) (0) 67.23 (+1) 64.04 (-4) 62.89 (-6) 71.56 (+7) 60.83 (-9) 61.76 (-8) 66.89 35/12467,6/124 D/W*35/12467 SLA 393.4 382.7 369.4 367.8 386.6 402.4 402.9 372.1* 409.6 385.8 384.2 380.5 379.0 405.0 337.5 362.0 381.2 378.4 .356.6 369.8 373.9 D/W*4/12 (CM2 G-i) (-2) (-5) (-8) (-9) (-4) (0) (-8) (+1) (-5) (-5) (-6) (-6) ; (-io) (-3) (+19) (+1) (-5) (-1) HEIGHT 42.0 36.7 38.9 40.1 41.5 41.9 38.9 47.0 48.7 44.8 46.8 44.3* 45.5 50.1 50.5 49.3 47.5 50.2 47.6 50.6 49.9 35/12467 (CM) • ( + 8) (-6) (0) (+3) (+7) ( + 8) (-6) (-3) (-11) (-7) (-12) (-1) (+D (-1) (-5) (+1) (-5) (+1) STEM DIA. 5. 21 4.98 4.96 4.83 5.02 5. 05 5.08 5.23 5.43 5.14 5.35 5.27 5.45 5.21 5.62* 5.42 5.42 5.54 5.35 5.47 5.25 7/1246,FC/DW*l/2 (MM) ( + 3) (-2) (-2) (-5) (-1) (-1) (0) ( + 4) (-1) (+3) (+1) ( + 5) (+7) ( + 3) (+3) ( + 6) ( + 2) ( + 4) D/W*7/1246 90 Greenhouse-grown Phaseolvs vulgaris p l a n t s were s p r a y e d 3 t i m e s w i t h 5 e x t r a c t s o f Macrocystis integrifolia o b t a i n e d by t h e methods o f Rademacher and Graebe (1984). Each t r e a t m e n t (1 t o 5) c o n s i s t e d o f a f r a c t i o n d e s i g n e d t o s e p a r a t e d i f f e r e n t groups o f phytohormones. Treatment 6 was t h e u n f r a c t i o n a t e d l i q u e f i e d k e l p , and Treatment 7 was t h e c o n t r o l s p r a y o f t a p w a t e r . Wet t r e a t m e n t s were kept w a t e r l o g g e d , f i e l d c a p a c i t y t r e a t m e n t s a t -30 t o -50 kPa, and d r y t r e a t m e n t s a t -120 t o -150 kPa. A s t e r i s k s i n d i c a t e means s i g n i f i c a n t l y d i f f e r e n t from t h e c o n t r o l s w i t h i n each water p o t e n t i a l (p< 0.1). Numbers i n b r a c k e t s a r e p e r c e n t o f c o n t r o l means w i t h i n w a t e r p o t e n t i a l s . groups of means orig i n a t i n g with the stimulatory effects of Fraction 4 on wet s o i l and Fraction 2 in f i e l d capacity s o i l . This relationship was maintained for leaf fresh weight. Stem fresh weight also d i f f e r e d between these two groups, with Fractions 3+5 depressing t h i s y i e l d variable, primarily due to the eff e c t of Fraction 5 i n f i e l d capacity s o i l . The remaining fractions had l i t t l e e f f e c t on stem fresh weight, with the exception of Fraction 4, which contributed most heavily to the net pos i t i v e e f f e c t of Fractions 1+2+4+6+7. Fractions 3+5 had l i t t l e e f f e c t on number of pods, however Fractions 1 and 2 i n f i e l d capacity s o i l s and Fraction 4 in wet s o i l contributed to the s i g n i f i c a n t l y greater number of pods i n the group containing these means. In dry and f i e l d capacity s o i l s , a l l fractions had no or s l i g h t l y negative effects on SLA. Fraction 3, however, in wet s o i l conditions, increased the net eff e c t of Fractions 3+5 to a s i g n i f i c a n t l e v e l . F i n a l l y , the contrast between heights of these two groups of means was also s i g n i f i c a n t , primarily due to i n h i b i t i o n of stem elongation by both Fractions 3 and 5 i n f i e l d capacity and wet s o i l . In general, the main differences between these groups of means were primarily the result of s o i l moisture-specific stimulation by members of the 1+2+4+6+7 group (ie. leaf area, leaf fresh weight, stem fresh weight, number of pods), and to a lesser extent by dire c t i n h i b i t i o n by Fractions 3+5 (ie. LAR, height). The experiment-wide contrast between Fraction 7 and Fractions 1+2+4+6 also yielded s i g n i f i c a n t differences between means. This contrast compared the control plants, which were sprayed with water, to those fractions expected to contain the majority of phytohormonally active substances, which included the complete seaweed homogenate. Leaf dry weights were substantially increased by several f r a c t i o n s . The magnitude of the response was greatest for Fractions 1 and 2 i n f i e l d capacity s o i l , and Fractions 1 and 4 i n wet s o i l . Root weights were s i m i l a r l y affected, with Fractions 3 and 5 exerting l i t t l e e f f e c t , and Fractions 1, 4, and 6 i n f i e l d capacity s o i l and 2,4, and 6 i n wet s o i l contributing most to the increase i n root weight of t h i s group of means. The number of pods per pot was also affected d i f f e r e n t l y by the putative stimulatory fractions than by the control. Again Fractions 1 and 2 i n f i e l d capacity s o i l s and Fraction 4 in wet s o i l increased the magnitude of t h i s variable, "and contributed most to the difference between the grouped means. F i n a l l y stem diameter was s l i g h t l y but p o s i t i v e l y affected by the seaweed fractions in t h i s group compared to the controls. To summarize, the s i g n i f i c a n t main effects of Fractions 1+2+4+6 compared to the control was primarily the result of stimulation by Fractions 1 and 2 in f i e l d capacity s o i l and 92 by Fraction 4 i n wet s o i l . The t h i r d s i g n i f i c a n t main ef f e c t was the result of the contrast between Fraction 6, the complete seaweed homogenate, and Fractions 1+2+4, the fractions which should contain the bulk of the phytohormones. The only variable affected was LAR, which was greater for those plants sprayed with Fractions 1+2+4 i n dry and f i e l d capacity s o i l , where Fraction 6 had l i t t l e e f f e c t . In wet s o i l , i n h i b i t i o n by Fraction 6 and stimulation by Fraction 4 contributed to the significance of t h i s difference. Seven of the interactions between s o i l water potential and spray treatment were s i g n i f i c a n t for at least one variable. The FC vs D+W by Fraction 3+5 vs 1+2+4+6+7 was si g n i f i c a n t for stem dry weight and the number of pods per pot. This result suggests a d i f f e r e n t net plant response to • to the putative i n h i b i t o r y fractions and a l l other fractions on f i e l d capacity versus moisture-stressed s o i l . Stem dry weight was increased by Fractions 3+5 in f i e l d capacity s o i l , but had l i t t l e e f f e c t on either dry or wet s o i l . In comparison, the remaining fractions increased stem dry weight more in f i e l d capacity s o i l than i n the moisture-stressed s o i l s . The differences were thus opposite i n di r e c t i o n and of d i f f e r e n t magnitude i n f i e l d capacity compared to moisture-stress conditions. In addition the number of pods per pot was increased more by Fractions 1+2+4+6+7 i n f i e l d capacity s o i l than i t was in the moisture stressed s o i l . These groups of means also indicated a d i f f e r i n g response in wet as compared to dry s o i l , since the D vs W by Fractions 3+5 vs Fractions 1+2+4+6+7 was s i g n i f i c a n t . LAR of plants treated with Fractions 3+5 in dry s o i l was higher than i t was for the remaining sprays. In wet s o i l , however, the opposite occurred i n that Fractions 3+5 caused a decrease i n the LAR compared to the remaining f r a c t i o n s . Si m i l a r l y plants responded d i f f e r e n t l y to Fraction 7, the control, than to Fractions 1+2+4+6, depending on whether the plants were maintained i n dry or wet s o i l . This interaction between water potential and the control versus "stimulatory" fractions (D vs W by Fraction 7 vs Fractions 1+2+4+6) was si g n i f i c a n t for several variables. Results were sim i l a r for pod, shoot, and plant dry weights, with, the putative phytohormone-containing fractions increasing dry weights on wet s o i l , and having s l i g h t l y negative or no ef f e c t on dry s o i l . This relationship was also observed for harvest index. While stem diameter was unaffected by the seaweed fractions on dry s o i l , i t was s l i g h t l y increased by the putative stimulatory fractions, e s p e c i a l l y Fraction 1, on wet s o i l . The interaction between f i e l d capacity versus moisture-stressed s o i l water potentials and Fraction 4 versus Fractions 1+2 was s i g n i f i c a n t for stem weight, shoot dry weight, and number of pods. In f i e l d capacity s o i l , Fraction 4 decreased stem dry weight, while Fractions 1+2 increased i t . In s o i l with moisture stress applied, stem weight was increased by Fraction 4 more than by Fractions 1+2. This pattern was also evident for shoot dry weight and the number of pods per pot. With bean number, the magnitude of these differences was larger, with Fractions 1+2 increasing the number of pods by 30% above control l e v e l s , while Fraction 4 increased i t by only 3% on f i e l d capacity s o i l . The opposite occurred on stressed s o i l , with a 9.5% stimulation by Fraction 4, and e s s e n t i a l l y no e f f e c t from Fractions 1+2. The D vs W by Fraction 4 vs Fraction 1+2 was s i g n i f i c a n t for many y i e l d variables. Leaf area was s l i g h t l y decreased by Fraction 4 and Fractions 1+2 i n dry s o i l conditions, but on wet s o i l Fraction 4 greatly increased leaf area over Fractions 1+2, which remained at control l e v e l s . Stem fresh weight was negatively affected by Fraction 4 on dry s o i l , and Fractions 1+2 remained at control l e v e l s . This d i f f e r s markedly from t h e i r effects i n wet s o i l , where stem fresh •weight was increased 23% over control levels while Fractions 1+2 increased i t by only 5%. Shoot fresh weight followed t h i s trend i n wet s o i l , with a 19% increase caused by Fraction 4, while yields from Fraction 1+2 treated plants remained at control l e v e l s . Both treatments were s l i g h t l y i n h i b i t o r y on dry s o i l . Dry weight differences p a r a l l e l l e d these fresh weight 95 differences. Stem dry weight was negatively affected on dry s o i l by Fraction 4, and Fraction 1+2 resulted i n a s l i g h t increase above control values. On wet s o i l , Fraction 4 increased stem dry weight by 24% over the control, while Fraction 1+2 treated plants remained at control l e v e l s . Pod dry weights were s i m i l a r l y i n h i b i t e d by both treatments on dry s o i l , but on wet s o i l Fraction 4 stimulated pod dry weight accumulation to 30% beyond control plants and Fractions 1+2 increased pod dry weight by 7%. Root and plant dry weights were also stimulated on plants i n wet s o i l sprayed with Fraction 4 (40% and 14% respectively). Under t h i s moisture regime Fractions 1+2 stayed near control l e v e l s . Shoot/root r a t i o was increased by Fraction 4 on dry s o i l , but decreased by t h i s f r a c t i o n on wet s o i l . This contrasts with the effects of Fractions 1+2 on the shoot/root r a t i o . On dry s o i l Fractions 1+2 had l i t t l e e f f e c t , while on wet s o i l these fractions were i n h i b i t o r y . The interaction FC/D+W by 1/2 was s i g n i f i c a n t for pod number, harvest index and stem diameter. In s o i l maintained at f i e l d capacity, pod number was stimulated s i g n i f i c a n t l y by both Fraction 1 and 2. Under s o i l moisture stress conditions niether f r a c t i o n had an e f f e c t . Harvest index was increased by Fraction 2, but Fraction 1 had l i t t l e e f f e c t on plants i n f i e l d capacity s o i l . Both fractions were s l i g h t l y i n h i b i t o r y on moisture stressed s o i l , with Fraction 2 most i n h i b i t o r y . Stem diameter was unaffected by Fraction 1 i n f i e l d capacity s o i l , and Fraction 2 under moisture stress conditions. Fraction 2, however, increased stem diameter i n f i e l d capacity s o i l , while Fraction 1 increased i t under moisture stress conditions. The f i n a l s i g n i f i c a n t i nteraction indicated a d i f f e r i n g e f f e c t of Fraction 1 and 2 on shoot dry weight i n wet as compared to dry s o i l . Under dry s o i l conditions, both fractions caused a s l i g h t i n h i b i t i o n of shoot dry weight of sprayed plants. On wet s o i l , Fraction 1 was s l i g h t l y stimulatory, while Fraction 2 treated plants stayed at control l e v e l s . It i s evident from Table 5 that the eff e c t of the various seaweed treatments d i f f e r e d according to the moisture regime to which the plants were subjected. This e f f e c t i s r e f l e c t e d in the many s i g n i f i c a n t interactions between water pote n t i a l and spray treatments. When Duncan's Multiple Range test was applied to the fractions within each water potential treatment, many fractions, including those expected to contain stimulatory phytohormones were actually i n h i b i t o r y to many of the y i e l d variables. These negative effects were confined to the a e r i a l parts of the plants: root weights were not s i g n i f i c a n t l y affected by any of the sprays under dry or f i e l d capacity s o i l moisture conditions. Furthermore, i n dry 97 s o i l , negative effects were confined to a reduction i n pod dry weight. Since t h i s y i e l d component contributed the greatest proportion to shoot and plant dry weights, these y i e l d variables were also reduced. The reduction of pod dry weight i s also responsible for the s i g n i f i c a n t reduction i n harvest index caused in dry s o i l conditions. In s o i l s maintained at f i e l d capacity, the s i t u a t i o n was more complex, with some fractions i n h i b i t i n g some y i e l d variables, and others s i g n i f i c a n t l y increasing them over appropriate control l e v e l s . Those variables decreased s i g n i f i c a n t l y by spray treatments were reduced exclusively by those fractions which were not expected to contain major phytohormones, namely Fractions 3 and 5. In addition, those fractions which reduced y i e l d components affected only fresh weights: dry weights were unaffected. Variables thus affected were also confined to the a e r i a l parts of the plant. Leaf fresh weight, bean pod fresh weight, and shoot fresh weight were a l l reduced by the same magnitude by Fraction 3. Again shoot fresh weight was s i g n i f i c a n t l y reduced because t h i s variable i s comprised primarily of le a f and pod weights. Stem fresh weight was s i g n i f i c a n t l y reduced by Fraction 5. This reduction i s also r e f l e c t e d i n the s i g n i f i c a n t l y reduced height of plants sprayed with t h i s f r a c t i o n . One secondary variable, SLA, was reduced by a putative stimulatory f r a c t i o n , Fraction 1. SLA (leaf area/leaf dry weight), which 98 i s usually an indicator of leaf thickness, was reduced by a s i g n i f i c a n t increase i n leaf dry weight, since the leaf area remained unaltered. While the variables described above were reduced by some of the f o l i a r l y applied seaweed fractions, several others were increased. Fraction 2 caused an increase of 16% i n leaf area, and corresponding increases in leaf fresh (12%) and dry (16%) weights. Because the increases were proportional, SLA was unaffected. Leaf dry weight was also increased s i g n i f i c a n t l y by f r a c t i o n 1, expected to contain the main auxin-like substances. In general the indices of plant growth were increased by Fraction 2, the f r a c t i o n expected to contain the auxin conjugates, as well as some cytokinin conjugates and rarer g i b b e r e l l i n s . In addition to the p o s i t i v e effects of t h i s f r a c t i o n on leaf growth parameters, Fraction 2 increased pod dry weight by 15%. This increase was apparently the result of a substantial (40%) increase i n the number of pods per pot. Since these increases were disproportionate, average pod weight must have been reduced. In a longer term experiment, one would therefore expect further increases in pod dry weight as the increased number of smaller pods matured. The increased pod weight contributed to concommitant s i g n i f i c a n t increases i n shoot and plant dry weight, and was responsible for the s i g n i f i c a n t 9% increase i n harvest index. A further s i g n i f i c a n t e f f e c t of f o l i a r spray on plants i n f i e l d capacity s o i l was the increase i n plant dry weight caused by Fraction 6, the complete homogenate. This increase was due to the cumulative ef f e c t s of small, i n d i v i d u a l l y non-significant increases i n leaf, stem, pod and root dry weights. In wet s o i l , plants responded s i g n i f i c a n t l y to only one fract i o n , Fraction 4, with the exception of stem diameter, which was s i g n i f i c a n t l y increased by Fraction 1. Fraction 4, expected to contain the major cytokinins, caused sizable s i g n i f i c a n t increases in leaf area (22%), fresh (24%) and dry (19%) weights, stem fresh (23%) and dry (24%) weights, shoot fresh (19%) and dry (10%) weights, pod dry weight (30%), root dry weight (40%), and plant dry weight (14%). Number of pods per pot was also increased 25% by t h i s f r a c t i o n . None of the secondary variables (HI, SLA LAR) were s i g n i f i c a n t l y affected, i n d i c a t i n g that growth increases among y i e l d components were proportional. Overall, several points emerge from the analysis of t h i s experiment. The response to f o l i a r treatments was dependent on s o i l water p o t e n t i a l . While wet and dry weights of y i e l d components responded p o s i t i v e l y to only a single fr a c t i o n , Fraction 4 i n wet s o i l , i n f i e l d capacity s o i l both p o s i t i v e and negative responses were observed. Positive responses 100 were the result of applications of Fractions 1 and 2. Negative responses were the result of Fraction 3 and 5 application, and were confined to differences i n fresh weights: dry weights were unaffected compared to control levels when these fractions were applied. F i n a l l y i n dry s o i l a l l treatments resulted i n a negative effect on pod fresh and dry weights. This reduction was of a magnitude that led to s i g n i f i c a n t decreases i n other variables (ie,shoot and plant weights) of which pod weight was a component. Despite these soil-moisture s p e c i f i c differences i n response, several experiment-wide in d i v i d u a l degree of freedom comparisons were also s i g n i f i c a n t . The contrast between putative i n h i b i t o r y (Fractions 3+5) and non-inh i b i t o r y (Fractions 1+2+4+6+7) fractions was s i g n i f i c a n t for several variables including leaf area, l e a f fresh weight, number of pods and plant height. These differences, however, can be attributed to large increases r e s u l t i n g from a few treatments rather than smaller across the board increases gaining significance through the increased power of t h i s s t a t i s t i c a l approach. S i m i l a r l y the main effects for the control versus putative stimulatory fractions (Fraction 7 versus Fractions 1+2+4+6) i n leaf dry weight, root dry weight and number of pods were due to large contributions from a few treatments i n wet and f i e l d capacity s o i l rather than from a 101 more general but smaller e f f e c t . In the case of stem diameter, however, the significance of t h i s main effect was the result of the a b i l i t y of t h i s s t a t i s t i c a l approach to pick up small increases which were i n d i v i d u a l l y i n s i g n i f i c a n t , but generally d i s t r i b u t e d among the treatments. Although only one variable, plant dry weight i n f i e l d capacity s o i l , was s i g n i f i c a n t l y p o s i t i v e l y affected by the complete seaweed homogenate, substantial root weight increases (23%) were observed i n wet s o i l . This also contributed to the significance of the control versus Fraction 1+2+4+6 contrast for t h i s variable. The f a i l u r e of the complete homogenate to equal the po s i t i v e e f f e c t s of Fractions 1, 2, and 4 indicates that under these growing conditions the in h i b i t o r y fractions are s u f f i c i e n t l y powerful to overcome the stimulatory components, or that interactions among the stimulatory components of the homogenate negate each other. 102 3.2 Phytohormone Analysis Results of auxin, cytokinin and one g i b b e r e l l i n bioassay for samples of M. i n t e g r i f o l i a are presented below. Since these results indicated the highest auxin-like and cytokinin-l i k e substances were present i n kelp c o l l e c t e d i n May, further analyses were performed on these samples using additional extraction, p u r i f i c a t i o n and chromatographic techniques. 3.2.1 Analysis of Monthly Samples: Recovery of Standards: Recovery of IAA: Figure 4 represents the results of GLC (Section 2.4.3) of the auxin f r a c t i o n of an extract obtained according to Rademacher and Graebe (1984) (Fraction I, Figure 1, Section 2.4.2). Figure 4A shows the chromatogram of 1 u l . . -1 of stock solution containing 1 ug u l of IAA. The IAA peak at retention time (RT) 3.78 min. had a peak area of'121, 600, and a second peak of unknown o r i g i n at RT 4.49. This extraneous peak has a RT similar to that of indolepyruvic acid (not shown). Injection of a similar volume of the d e r i v i t i z e d extract of added IAA stock solution made up to i t s o r i g i n a l volume revealed a peak with a RT of 3.79 min. 103 w w o w w Q H b 4A 2.0 4.0 minutes 6.0 w w 2 O o< Ui w Pi Q H IK 4B T 2.0 4:0 minutes 6.1 Figure 4 Recovery of IAA Following Extraction and P u r i f i c a t i o n 4A: Chromatogram of authentic IAA d e r i v i t i z e d with BSTFA Peak 1: RT=3.78, Arfea=121600. 4B: Chromatogram of a s i m i l a r quantity of IAA following extraction according to Rademacher and Graebe (1984). Fraction I was dried for analysis and d e r i v i t i z e d with BSTFA. Peak 1: RT=3.79, Area=98360. GLC Conditions: S i l a n i z e d glass column (2mmx6') of 3% SE on GasChrome Q; N9 flow rate 29 ml m i n - 1 ;FID temp. 350° i n j e c t o r temp. 250°C; oven temp. 185°C, 2 min., 30°C min to 235°C, 235°C, 40 min.. IAA= indoleacetic acid. 104 and a peak area of 98,360 (Figure 4B). The r a t i o of the peak areas (98,360/121,600) indicates a recovery of 58% of the IAA o r i g i n a l l y present p r i o r to extraction. The recovery of IAA from TLC plates run as described i n Section 2.4.3 was estimated i n the same manner. Results are presented i n Figure 5. IAA peaks were observed i n Rf zones 0.5 and 0.6 (Figure 5B and Figure 5C). Comparison of the sum of the peak areas at RT 3.7 9 min. with that of the o r i g i n a l chromatographed stock solution (Figure 5A) indicates a recovery of 17%. Overall recovery of IAA was therefore estimated as the product of the recoveries from extraction and p u r i f i c a t i o n (58%), and chromatography (17%), which indicates a 10% recovery of I7AA for the combined processes. Recovery of Cytokinins: Cytokinin recoveries were estimated in an analogous fashion and are presented i n Figure 6. Figure 6A represents a chromatogram of each cytokinin standard (ie. the o r i g i n a l stock solution). Figure 6B shows a chromatogram of the cytokinin-containing extract components. By comparing standard stock solution to extract peak areas, recoveries for isopentenyl adenine (RT 3.31 min.) and isopentenyl adenosine (RT 10.39 min.) were 36% and 75% respectively. Figure 7 shows the recovery of these compounds a from Dowex 50X8 column as described i n Section 2.4.3. 7A 105 w w 55 O w w Pi p H td (0 2 O Ch w w Pi O H tl4 T T 2-0 4.0 minutes w Ui 55 O CU Ui w Pi 1 1— 2.0 4.0 minutes Figure 5 2.0 4.0 minutes Recovery of IAA Following Thin Layer Chromatography 5A: Chromatogram of authentic IAA d e r i v i t i z e d with BSTFA. Peak 1: RT=3.80, Area=245400. 5B,C: Chromatogram of a s i m i l a r quantity of IAA following TLC and e l u t i o n from Rf 0.5 (5B: Peak 1; RT=3.81, Area=21760) and 0.6 (5C: Peak 1; RT=3.81, Area=20200). TLC Conditions: IAA applied to Kieselgel F254 plates i n methanol and run i n isopropanol/ammonia/water (10 / l / l : v / v / v ) . GLC Conditions: S i l a n i z e d glass column (2mmx6') of 3% SE-30 on GasChrome Q; N o flow rate 29 ml m i n - 1 ; FID temp. 350°C; i n j e c t o r temp. 250°C; oven temp. 185°C, 2 min., 30 bC min. 1 to 235°C, 235°C, 40 min.. IAA=indoleacetic acid. 106 w w 2 O W w & o H (14 Id OT O 04 OT W Ci 2.0 4.0 6.0 8.0 minutes LO.O 12. 2.0 4.0 6.0 8.0 minutes LO.O 12.0 Figure 6 Recovery of Isopentenyl Adenine (IPA) and Isopentenyl adenosine (IPAR) Following Extraction and P u r i f i c a t i o n 6A: Chromatogram of authentic standards d e r i v i t i z e d with BSTFA. Peak 1: IPA; RT=3.31, Area=637100. Peak 2: IPAR; RT=10.39, Area=119200. 6B: Chromatogram of a s i m i l a r quantity of standards following extraction according to Rademacher and Graebe (1984) . The butanolic Fraction IV was dried for analysis and d e r i v i t i z e d with BSTFA. Peak 1: IPA; RT=3.37, Area=235300. Peak 2: IPAR; RT=10.55, Area=89860. GLC Conditions: S i l a n i z e d glass column (2mmx6') of 3% SE-30 on GasChrome Q ; N o flow rate 29 ml m i n - 1 ;FID temp. 350°C; i n j e c t o r temp. 25u°C; oven temp. 185°C, 2 min., 30°C min to 235°C, 235°C, 40 min.. -1 107 m W SS O CK w Id Oi a H minutes I 1 I 1 T 1 2.0 4.0 6.0 8.0 l6.0 l i minutes 7A Figure 7 7B Recovery of Isopentenyl Adenine and Isopentenyl adenosine Following Chromatography on Dowex 50X 7A: Chromatogram of authentic standards d e r i v i t i z e d with BSTFA. 7B: Chromatogram of a sim i l a r quantity of standards following application to a column of Dowex 50X (2cm x 15cm) (Stoddart et a l . , 1984). Tha ammoniacal eluate was dried for analysis and d e r i v i t i z e d with BSTFA. GLC Conditions: Si l a n i z e d glass column (2mmx6') of 3% SE-30 on GasChrome Q; N? flow rate 2 9 ml m i n - 1 ;FID temp. 350°C; i n j e c t o r temp. 25D°C; oven temp. 185°C, 2 min., 30°C min to 235°C, 235°C, 40 min.. -1 108 shows the standard, 7B shows the standard recovered from the column. By c a l c u l a t i n g peak area ra t i o s the recovery of these compounds was calculated as 93% and 58% for isopentenyl adenine and isopentenyl adenosine respectively. Thus, the product of the extraction and chromatographic recoveries yields 33% recovery for isopentenyl adenine and 44% recovery for isopentenyl adenosine. Recovery from TLC was not evaluated. Phytohormone A c t i v i t y Estimates: Estimates of auxin-like and c y t o k i n i n - l i k e b i o l o g i c a l a c t i v i t y were made based on bioassay responses (Section 2.4.1) of chromatographed extracts of the appropriate fractions generated by the methods of Rademacher and Graebe (1984). Estimates generated for each were corrected for losses during the procedure according to recoveries calculated above. Auxin Bioassays: Results of the Avena hypocotyl elongation bioassay for auxins (Section 2.4.1) are presented i n Table 6. Fractions were chromatographed p r i o r to bioassay as described in Section 2.4.3. Data presented represent the mean of two determinations and have been corrected for losses as described above. Figures 8 and 9 show the bioassay results for two samples of seaweed c o l l e c t e d i n May, Figures 10 and 11 the bioassay results for two samples of seaweed co l l e c t e d 109 MONTH OF KELP COLLECTION AUXIN-LIKE ACTIVITY (ug IAA 100 g 1 FW) S .E. MAY 193.0 133 .0 JULY 8.0 5 .0 SEPTEMBER 1.1 0 .1 TABLE 6 Auxin-like A c t i v i t y of Extracts of Macrocystis integrifolia Tissue samples were macerated and extracted into an a c i d i c ethyl acetate phase containing the auxins (Fraction I of Rademacher and Graebe (1984)), which was then chromatographed on Kieselgel F254 TLC plates run i n isobutanol/ammonia/water (10/1/1:v/v/v). Rf zones were eluted p r i o r to bioassay using the Avena straight growth bioassay (Nitsch and Nitsch, 1956). Auxin-like a c t i v i t y of Rf zones s i g n i f i c a n t l y greater than the control (LSD, p< 0.05) was converted to indoleacetic acid (IAA) equivalents per 100 g tissue by substitution into the regression equation for standard concentrations of IAA assayed concurrently, and summed. 110 AUXIN BIOASSAY 100 GM MAY SEAWEED 0 -7 -6 -5 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 LOG M IAA Rf Figure 8 Auxin bioassay of extract of 100 g May seaweed Results of Avena straight growth auxin bioassay (Nitsch and Nitsch, 1956) of an a c i d i c ethyl acetate-soluble phytohormone f r a c t i o n (Fraction I) of M. i n t e g r i f o l i a obtained by the methods of Rademacher and Graebe (1984). The f r a c t i o n was dried and applied to Kieselgel plates which were developed i n isopropanol/ ammonia/water (10/1/1 :v/v/v). . Horizontal bars represent Rf of authentic standard. Asterisks indicate a c t i v i t y s i g n i f i c a n t l y greater than the control (LSD, p<0.05). IAA = indoleacetic acid. I l l AUXIN BIOASSAY 0 -7 -6 -5 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 LOG M IAA Rf Figure 9 Auxin bioassay of extract of 100 g May seaweed Results of Avena straight growth auxin bioassay (Nitsch and Nitsch, 1956) of an a c i d i c ethyl acetate-soluble phytohormone f r a c t i o n (Fraction I) of M. i n t e g r i f o l i a obtained by the methods of Rademacher and Graebe (1984). The f r a c t i o n was dried and applied to Kies e l g e l plates which were developed i n isopropanol/ ammonia/water (10/1/1:v/v/v). Horizontal bars represent Rf of authentic standard. Asterisks indicate a c t i v i t y s i g n i f i c a n t l y greater than the control (LSD, p<0.05). IAA = indoleacetic acid. 112 AUXIN BIOASSAY 100 GM JULY SEAWEED IAA 4 wk ' I 1 0 -7 -6 -5 LOG M IAA 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Rf Figure 10 Auxin bioassay of extract of 100 g July seaweed Results of Avena straight growth auxin bioassay (Nitsch and Nitsch, 1956) of an a c i d i c ethyl acetate-soluble phytohormone f r a c t i o n (Fraction I) of M. i n t e g r i f o l i a obtained by the methods of Rademacher and Graebe (1984). The f r a c t i o n was dried and applied to Kieselgel plates which were developed i n isopropanol/ ammonia/water (10/1/1:v/v/v). Horizontal bars represent Rf of authentic standard. Asterisks indicate a c t i v i t y s i g n i f i c a n t l y greater than the control (LSD, p<0.05). IAA = indoleacetic acid. 113 AUXIN BIOASSAY 0 -7 -6 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 LOG M IAA Rf Figure 11 Auxin bioassay of extract of 100 g July seaweed Results of Avena straight growth auxin bioassay (Nitsch and Nitsch, 1956) of an a c i d i c ethyl acetate-soluble phytohormone f r a c t i o n (Fraction I) of M. i n t e g r i f o l i a obtained by the methods of Rademacher and Graebe (1984). The f r a c t i o n was dried and applied to Kieselgel plates which were developed i n isopropanol/ ammonia/water (10/1/1:v/v/v). Horizontal bars represent Rf of authentic standard. Asterisks indicate a c t i v i t y s i g n i f i c a n t l y greater than the control (LSD, p<0.05). IAA = indoleacetic acid. 114 AUXIN BIOASSAY 0 -7 -6 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 LOG M IAA Rf Figure 12 Auxin bioassay of extract of 100 g September seaweed Results of Avena straight growth auxin bioassay (Nitsch and Nitsch, 1956) of an a c i d i c ethyl acetate-soluble phytohormone f r a c t i o n (Fraction I) of M. integrifolia obtained by the methods of Rademacher and Graebe (1984). The f r a c t i o n was dried and applied to Kieselgel plates which were developed i n isopropanol/ ammonia/water (10/1/1:v/v/v). Horizontal bars represent Rf of authentic standard. Asterisks indicate a c t i v i t y s i g n i f i c a n t l y greater than the control (LSD, p<0.05). IAA = indoleacetic acid. 115 AUXIN BIOASSAY 0 -7 -6 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 LOG M IAA Rf Figure 13 Auxin bioassay of e x t r a c t of 100 g September seaweed Re s u l t s of Avena s t r a i g h t growth auxin bioassay (Nitsch and N i t s c h , 1956) of an a c i d i c e t h y l a c e t a t e - s o l u b l e phytohormone f r a c t i o n ( F r a c t i o n I) of M. integrifolia obtained by the methods of Rademacher and Graebe (1984). The f r a c t i o n was d r i e d and a p p l i e d to K i e s e l g e l p l a t e s which were developed i n iso p r o p a n o l / ammonia/water (10/1/1:v/v/v). H o r i z o n t a l bars represent Rf of authentic standard. A s t e r i s k s i n d i c a t e a c t i v i t y s i g n i f i c a n t l y g reater than the c o n t r o l (LSD, p<0.05). IAA = i n d o l e a c e t i c a c i d . 116 in July, and Figures 12 and 13 the bioassay results for two samples of seaweed co l l e c t e d i n September. Most of the auxin-like a c t i v i t y shown in Figure 8 was found i n RF zone 0.5, the location of authentic IAA i n t h i s chromatographic system. Additional a c t i v i t y was seen i n Rf zones 0.8 and 0.9. The o r i g i n of t h i s a c t i v i t y may have been IAA precursors or conjugates metabolized to t h e i r active forms by the bioassay tissues, or from auxin-active substances other than indole derivatives. The second assessment (Figure 9), showed auxin a c t i v i t y at the o r i g i n , at Rf 0.3, and at Rf zone 0.8. Retention of a c t i v i t y at the o r i g i n , and the possible retardation of mobility of IAA to Rf 0.3 could indicate poor loading of the plate, or overloading of the plate. In addition, the much lower y i e l d i n t h i s assay compared to the previous one could be an i n d i c a t i o n that the separation of auxin-like substances from possible anti-auxins or i n h i b i t o r s was impaired as well. A l t e r n a t i v e l y the a c t i v i t y at Rf 0.3 could be the result of breakdown of IAA into less mobile precursors or derivatives with less mobility, followed by re-metabolism to t h e i r active forms by the bioassay tissues. Figure 10 shows poor resolution of auxin-like substances from seaweed co l l e c t e d i n July, since a c t i v i t y was seen i n Rf zones 0.3 to 1.0. Most a c t i v i t y was observed i n Rf zone 0.5, 117 where authentic IAA would be expected. Again a c t i v i t y peaks were observed in zones 0.8 and 0.9. The second assay, shown in Figure 11, has auxin-like a c t i v i t y confined to Rf zones 0.5 and 0.6, where authentic IAA would be expected. Bioassay of the auxin f r a c t i o n from seaweed co l l e c t e d i n September i s shown in Figure 12. A c t i v i t y was observed only in the Rf zone i n which authentic IAA i s found. The second assessment (Figure 13), however, had additional peaks at Rf 0.3 and 0.6. In summary, although the v a r i a t i o n i n t o t a l auxin-like a c t i v i t y between duplicate assays was high, a decreasing l e v e l of auxin-like a c t i v i t y i n the kelp plants as the growing season progressed was substantial. Auxin-like a c t i v i t y was highest in plants c o l l e c t e d i n May, lower i n kelp c o l l e c t e d i n July, and had declined to very low l e v e l s by September. Cytokinin Bioassays: Results of the Amaranthus cytokinin bioassay (Section 2.4.1) of the cytokinin f r a c t i o n obtained as described in Section 2.4.2 (Figure 1, Fraction IV) and subjected to TLC as described i n Section 2.4.3 are presented in Table 7. Data represent the mean of two determinations for seaweed co l l e c t e d i n May, July and September. Correction for losses during extraction and ion exchange chromatography have been made using the correction factors calculated above. For cytokinin-active substances with r e l a t i v e chromatographic 118 MONTH OF KELP CYTOKININ-LIKE S.E, COLLECTION ACTIVITY (ug IPA 100 g" 1 FW) MAY 0.80 0. 10 JULY 0.29 0. 15 SEPTEMBER 0.03 0. 02 TABLE 7 Cytokinin-like A c t i v i t y of Extracts of Macrocystis integrifolia. Tissue samples were macerated and extracted into a butanolic phase containing the cytokinins (Fraction IV of Rademacher and Graebe (1984)), which was then chromatographed an a Dowex 50X ion exchange column (Van Staden,1976). The ammoniacal eluate was dried, and the residue chromatographed on Kieselgel F254 TLC plates run i n isobutanol/ammonia/water (10/1/1:v/v/v). Rf zones were eluted wth methanol p r i o r to bioassay using the Amaranthus cytokinin bioassay (Biddington and Thomas, 1973) . Cytokinin-like a c t i v i t y of Rf zones s i g n i f i c a n t l y greater than the control (LSD, p< 0.05) was converted to isopentenyl adenine (IPA) equivalents per 100 g tissue by substitution into the regression equation for standard concentrations of IPA assayed concurrently, and summed 119 m o b i l i t i e s similar to those of r i b o s y l cytokinins, the correction factor for isopentenyl adenosine was used. Those substances with r e l a t i v e chromatographic m o b i l i t i e s similar to those of cytokinin bases were corrected using the factor calculated for isopentenyl adenine. Bioassay results for each determination are presented i n Figures 14 to 19. Figure 14 shows three zones possessing c y t o k i n i n - l i k e a c t i v i t y from seaweed co l l e c t e d i n May at Rf zones 0.4, 0.7, 0.8 and 0.9. Authentic zeatin riboside chromatographs at Rf 0.4, isopentenyl adenosine at Rf 0.7-0.8, and isopentenyl adenine at Rf 0.8-0.9. Figure 15 shows a very s i m i l a r a c t i v i t y p r o f i l e , with peaks i n a c t i v i t y at Rf 0.4, 0.7, and 0.8. Seaweed c o l l e c t e d i n July (Figure 16) showed cytokinin-l i k e a c t i v i t y at Rf 0.6, which does not correspond to any available standard, however the second assessment, shown in Figure 17, indicates b i o l o g i c a l a c t i v i t y at Rf 0.3 and 0.4 (zeatin riboside), 0.5 (zeatin) and 0.7 (isopentenyl adenosine). The active region (Rf 0.6) was corrected using the correction factor for isopentenyl adenine, since slower mobility due to overloading or i n e q u i l i b r a t i o n i s more l i k e l y than higher mobility. Assessments of c y t o k i n i n - l i k e a c t i v i t y i n seaweed 120 CYTOKININ BIOASSAY 0 -8 -7 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 LOG M IPA Rf Figure 14 Cytokinin bioassay of extract of 100 g May seaweed Results of Amaranthus betacyanin cytokinin bioassay (Biddington and Thomas, 1973) of a butanolic phytohormone f r a c t i o n (Fraction IV) of M. i n t e g r i f o l i a obtained by the methods of Rademacher and Graebe (1984). The f r a c t i o n was p r e - p u r i f i e d on a Dowex 50X column (2cm x 15 cm), (Van Staden, 1976) and the ammoniacal eluate dried and applied to Kie s e l g e l plates which were developed i n chloroform/methanol (17/3/:v/v). Rf zones were eluted with methanol p r i o r to bioassay. Horizontal bars represent Rf of authentic standard. Asterisks indicate a c t i v i t y s i g n i f i c a n t l y greater than the control (LSD, p<0.05). Z= zeatin, ZR= zeatin riboside, IPA= isopentenyl adenine, IPAR= isopentenyl adenosine. 121 CYTOKININ BIOASSAY 0 -9 -8 -7 -6 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 LOG M IPA Rf Figure 15 Cytokinin bioassay of extract of 100 g May seaweed Results of Amaranthus betacyanin cytokinin bioassay (Biddington and Thomas, 1973) of a butanolic phytohormone f r a c t i o n (Fraction IV) of M. i n t e g r i f o l i a obtained by the methods of Rademacher and Graebe (1984). The f r a c t i o n was p r e - p u r i f i e d on a Dowex 50X column (2cm x 15 cm), (Van Staden, 1976) and the ammoniacal eluate dried and applied to Kies e l g e l plates which were developed in chloroform/methanol (17/3/:v/v). Rf zones were eluted with methanol p r i o r to bioassay. Horizontal bars represent Rf of authentic standard. Asterisks indicate a c t i v i t y s i g n i f i c a n t l y greater than the control (LSD, p<0.05). Z= zeatin, ZR= zeatin riboside, IPA= isopentenyl adenine, IPAR= isopentenyl adenosine. 122 CYTOKININ BIOASSAY 0 -8 -7 -6 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 LOG M IPA Rf Figure 16 Cytokinin bioassay of extract of 100 g July seaweed Results of Amaranthus betacyanin cytokinin bioassay (Biddington and Thomas, 1973) of a butanolic phytohormone f r a c t i o n (Fraction IV) of M. i n t e g r i f o l i a obtained by the methods of Rademacher and Graebe (1984). The f r a c t i o n was p r e - p u r i f i e d on a Dowex 50X column (2cm x 15 cm), (Van Staden, 1976) and the ammoniacal eluate dried and applied to Kie s e l g e l plates which were developed i n chloroform/methanol (17/3/:v/v). Rf zones were eluted with methanol p r i o r to bioassay. Horizontal bars represent Rf of authentic standard. Asterisks indicate a c t i v i t y s i g n i f i c a n t l y greater than the control (LSD, p<0.05). Z= zeatin, ZR= zeatin riboside, IPA= isopentenyl adenine, IPAR= isopentenyl adenosine. 123 CYTOKININ BIOASSAY Figure 17 Cytokinin bioassay of extract of 100 g July seaweed Results of Amaranthus betacyanin cytokinin bioassay (Biddington and Thomas, 1973) of a butanolic phytohormone f r a c t i o n (Fraction IV) of M. i n t e g r i f o l i a obtained by the methods of Rademacher and Graebe (1984). The f r a c t i o n was p r e - p u r i f i e d on a Dowex 50X column (2cm x 15 cm), (Van Staden, 1976) and the ammoniacal eluate dried and applied to Ki e s e l g e l plates which were developed in chloroform/methanol (17/3/:v/v). Rf zones were eluted with methanol p r i o r to bioassay. Horizontal bars represent Rf of authentic standard. Asterisks indicate a c t i v i t y s i g n i f i c a n t l y greater than the control (LSD, p<0.05). Z= zeatin, ZR= zeatin riboside, IPA= isopentenyl adenine, IPAR= isopentenyl adenosine. 124 CYTOKININ BIOASSAY O -7 -6 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 LOG M IPA Rf Figure 18 Cytokinin bioassay of extract of 100 g September seaweed Results of Amaranthus betacyanin cytokinin bioassay (Biddington and Thomas, 1973) of a butanolic phytohormone f r a c t i o n (Fraction IV) of M. i n t e g r i f o l i a obtained by the methods of Rademacher and Graebe (1984). The f r a c t i o n was p r e - p u r i f i e d on a Dowex 50X column (2cm x 15 cm), (Van Staden, 1976) and the ammoniacal eluate dried and applied to Ki e s e l g e l plates which were developed i n chloroform/methanol (17/3/:v/v). Rf zones were eluted with methanol p r i o r to bioassay. Horizontal bars represent Rf of authentic standard. Asterisks indicate a c t i v i t y s i g n i f i c a n t l y greater than the control (LSD, p<0.05). Z= zeatin, ZR= zeatin riboside, IPA= isopentenyl adenine, IPAR= isopentenyl adenosine. 125 CYTOKININ BIOASSAY O -8 -7 -6 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 LOG M IPA Rf Figure 19 Cytokinin bioassay of extract of 100 g September seaweed Results of Amaranthus betacyanin cytokinin bioassay (Biddington and Thomas, 1973) of a butanolic phytohormone f r a c t i o n (Fraction IV) of M. integrifolia obtained by the methods of Rademacher and Graebe (1984). The f r a c t i o n was p r e - p u r i f i e d on a Dowex 50X column (2cm x 15 cm), (Van Staden, 1976) and the ammoniacal eluate dried and applied to Kie s e l g e l plates which were developed i n chloroform/methanol (17/3/:v/v). Rf zones were eluted with methanol p r i o r to bioassay. Horizontal bars represent Rf of authentic standard. Asterisks indicate a c t i v i t y s i g n i f i c a n t l y greater than the control (LSD, p<0.05). Z= zeatin, ZR= zeatin riboside, IPA= isopentenyl adenine, IPAR= isopentenyl adenosine. 126 c o l l e c t e d in September are i l l u s t r a t e d i n Figures 18 and 19. Cytokinin-active material was detected i n only one of the assays (Figure 18) at Rf zone 0.7. This corresponds to the mobility of isopentenyl adenosine i n t h i s assay system. In summary a trend i s evident i n Table 7 which indicates a decrease i n cy t o k i n i n - l i k e substances from May through July to September, when cytokinin a c t i v i t y approached n i l . G i b b e r e l l i n bioassay: A single g i b b e r e l l i n bioassay was performed on the g i b b e r e l l i n f r a c t i o n of seaweed co l l e c t e d i n May, obtained by the methods of Mukherji and Waring (1983). Results of the bioassay following paper chromatography as described i n Section 2.4.3 are presented i n Figure 20. An extraction e f f i c i e n c y of 40%, calculated by the extraction of a measured quantity of GA^ from water by the same method was used to y i e l d an estimate of 10 ug GA^ equivalents per 100 gm fresh weight of May seaweed (data not shown). Si g n i f i c a n t g i b b e r e l l i n - l i k e a c t i v i t y was detected at Rf zones 0.4, 0.5, and 0.6, with maximum a c t i v i t y at Rf 0.5. This corresponds to the mobility of authentic Ga^ established as described i n Section 2.4.3. 127 GIBBERELLIN BIOASSAY 0 O.l 1 10 100 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 MG L"1 GA3 Rf Figure 20 G i b b e r e l l i n bioassay of extract of 100 g May seaweed Results of Tan-ginbozu g i b b e r e l l i n bioassay (Murakami, 1968) of an a c i d i c ethyl acetate-soluble phytohormone f r a c t i o n of M. i n t e g r i f o l i a , obtained by the methods of Mukherji and Waring (1983). The extract was applied to Whatman 3 MM chromatography paper which was developed i n isopropanol/ ammonia/water (10/1/1:v/v/v), and each Rf zone was eluted with 1 ml 50% (v/v) aqueous acetone. Horizontal bars represent Rf of authentic GA3. Asterisks indicate a c t i v i t y s i g n i f i c a n t l y greater than the control (LSD, p<0.05). GA3 = g i b b e r e l l i c acid. 128 3.2.2 Additional Analysis of Seaweed Collected i n May: Use of the Methods of Taylor et a l . (1982): The methods of Taylor e_t_ a l . (1982) (Figure 2) were used to extract and p u r i f y c y t o k i n i n - l i k e substances from 5 gm of seaweed colle c t e d i n May. The n-butanol-soluble substances from the ammoniacal eluate of the c e l l u l o s e phosphate column were dried and taken up i n 2 ml 35% (v/v) aqueous EtOH for application to the Sephadex LH-20 column as described i n Section 2.4.3. Forty 20 ml fractions were collected, divided i n half, and assayed for cytokinin a c t i v i t y i n the soybean cal l u s bioassay (Section 2.4.1). Results are presented i n Figure 21. Five areas of cytokinin a c t i v i t y were observed. The f i r s t occupied a large zone immediately following the void volume. This elution volume did not correspond to that of any available standards, however l i t e r a t u r e reports indicate elution of cytokinin glucosides by less than 1 column volume of solvent (Horgan, 1978). The second peak of a c t i v i t y at Fraction 15 did not correspond to any available standards, and no l i t e r a t u r e reports any known cytokinin e l u t i n g at t h i s r e l a t i v e p o s i t i o n . The a c t i v i t y i n Fraction 21 corresponds to the elution volume of zeatin riboside, while that at Fractions 23 and 24 correspond to the e l u t i o n volume of zeatin. The f i n a l a c t i v i t y peak centered at Fraction 30, 129 CYTOKININ BIOASSAY 5 GM SEAWEED O-B-6 1 5 10 15 20 25 30 35 LOG M IPA FRACTION NUMBER Figure 21 Cytokinin bioassay of extract of 5 g May seaweed Results of soybean c a l l u s growth cytokinin bioassay (Miller, 1965) of an n-butanolic-soluble phytohormone f r a c t i o n of M. i n t e g r i f o l i a obtained by the methods of Taylor et al. (1982). The f r a c t i o n was applied to a c e l l u l o s e phosphate column (2 cm x 15 cm) and the ammoniacal eluate was concentrated and applied to a Sephadex LH 20 column (2 cm x 30 cm) eluted with 35% (v/v) aqueous ethanol. Twenty ml fractio n s were c o l l e c t e d for bioassay. Asterisks indicate a c t i v i t y s i g n i f i c a n t l y greater than the control (LSD, p<0.05). Horizontal bars indicate e l u t i o n volume of authentic standards. Z = zeatin, ZR = zeatin riboside, IPA = isopentenyl adenine, IPAR = isopentenyl adenosine 130 eluted at the same volume as isopentenyl adenine and isopentenyl adenosine. Calculation of the quantity of cytokinin a c t i v i t y i n isopentenyl adenine equivalents yields an uncorrected estimate of 27.9 ug 100 gm fresh weight 1, over 90% of which was l o c a l i z e d at the early eluting peak i n b i o l o g i c a l a c t i v i t y . Hydrolysis of Cytokinin Fraction Pr i o r to Bioassay: In a simi l a r experiment (Figure 22), 10 gm of tissue was extracted and chromatographed on ce l l u l o s e phosphate, however i n t h i s case the ammoniacal eluate was taken to dryness by rotary evaporation, then taken up i n 25 ml 0.6 M t r i f l u o r o a c e t i c acid. This was then heated for 3 hrs at 95°C p r i o r to rotary evaporation to dryness. The residue was taken up i n 35% (v/v) aqueous EtOH and applied to the Sephadex LH-20 column. This procedure' hydrolyses any glucosidic bonds and should free the parent molecule r e s u l t i n g i n a s h i f t i n a c t i v i t y to the retention volume of the parent molecule (Schliemann and Liebisch, 1984). Although the t o t a l calculated cytokinin -1 a c t i v i t y for the extract (10.6 ug 100 gm fresh weight ) was less than that calculated for the previous extraction (27.9 -1 ug 100 gm ), some a c t i v i t y was sh i f t e d from the early-eluting peak to the peak at Fraction 15-16. In the cyt o k i n i n - l i k e a c t i v i t y determination of unhydrolyzed cytokinin extract, 92% of the cyt o k i n i n - l i k e a c t i v i t y CYTOKININ BIOASSAY OF 10 GM MAY SEAWEED 0-9-B-7-6-5 1 5 10 15 20 25 30 35 40 LOG M IPA FRACTION NUMBER F i g u r e 22 C y t o k i n i n b i o a s s a y o f t r i f l o u r o a c e t i c a c i d - h y d r o l i z e d e x t r a c t o f 10 g May se a w e e d R e s u l t s o f s o y b e a n c a l l u s g r o w t h c y t o k i n i n b i o a s s a y ( M i l l e r , 1965) o f an n - b u t a n o l i c - s o l u b l e p h y t o h o r m o n e f r a c t i o n o f M. i n t e g r i f o l i a o b t a i n e d b y t h e m e t h o d s o f T a y l o r e t al. ( 1 9 8 2 ) . The f r a c t i o n was a p p l i e d t o a c e l l u l o s e p h o s p h a t e c o l u m n (2 cm x 15 cm) a n d t h e a m m o n i a c a l e l u a t e was c o n c e n t r a t e d , h y d r o l i z e d w i t h 0.6N t r i f l o u r o a c e t i c a c i d (3 h r , 100°C) ( S c h l i e m a n n a n d L i e b i s c h , 1 9 8 4 ) , a n d a p p l i e d t o a Se p h a d e x LH 20 c o l u m n (2 cm x 30 cm) e l u t e d w i t h 3 5 % (v/v) a q u e o u s e t h a n o l . Twenty m l f r a c t i o n s w e re c o l l e c t e d f o r b i o a s s a y . A s t e r i s k s i n d i c a t e a c t i v i t y s i g n i f i c a n t l y g r e a t e r t h a n t h e c o n t r o l (LSD, p < 0 . 0 5 ) . H o r i z o n t a l b a r s i n d i c a t e e l u t i o n v o l u m e o f a u t h e n t i c s t a n d a r d s . Z = z e a t i n , ZR = z e a t i n r i b o s i d e , I P A = i s o p e n t e n y l a d e n i n e , IPAR = i s o p e n t e n y l a d e n o s i n e 132 occurred i n the ear l y - e l u t i n g peak, whereas following hydrolysis 72% of the cy t o k i n i n - l i k e a c t i v i t y remained i n t h i s area. This indicates the possible presence of some cytokinin glucosyl derivatives. Analysis of 40, 10, and 1 g Seaweed: Since the previous experiments indicated less b i o l o g i c a l a c t i v i t y r e s u l t i n g from an assay of 10 gm of tissue than from 5 gm of tissue, an experiment was conducted to determine the relationship between the amount of seaweed extract assayed and the estimate of cytokinin a c t i v i t y r e s u l t i n g from that assay. N-butanolic extracts of c e l l u l o s e phophate column eluates (Figure 2) were streaked onto Whatman 3MM chromatography paper i n amounts equivalent to 1, 10, and 40 gm of o r i g i n a l seaweed fresh weight. These were developed i n isobutanol/25% ammonia/H^O (10/1/1:v/v/v), arid cut into Rf zones which were then included i n the soybean cal l u s bioassay (section 2.4.1). Results are presented i n Figure 23. -1 Calculation of isopentenyl adenine equivalents 100 gm -1 seaweed indicates 14 ug 100 gm i f 40 gm of seaweed are -1 assayed, 70 ug 100 gm i f 10 gm of seaweed are assayed, and -1 170 ug 100 gm i f 1 gm of seaweed i s assayed. This indicates the presence i n the extract of an anti-cytokinin or cytokinin i n h i b i t o r that has extraction, p a r t i t i o n , and CYTOKININ BIOASSAY o-g-B-7-6 0.1 0.5 1 0.1 0.5 1 0.1 0.5 1 LOG M IPA Rf Rf Rf Figure 23 Cytokinin bioassay of extract of 1, 10, and 40 g May seaweed Results of soybean c a l l u s growth cytokinin bioassay (Miller, 1965) of an n-butanolic-soluble phytohormone f r a c t i o n of M. i n t e g r i f o l i a obtained by the methods of Taylor et al. (1982). The f r a c t i o n was applied to a c e l l u l o s e phosphate column (2 cm x 15 cm) and the ammoniacal eluate was concentrated and applied to Whatman 3 MM chromatography paper which was developed i n isopropanol/ammonia/water (10/1/1:v/v/v). Chromatograms were cut into 10 Rf zones which were included i n the bioassay f l a s k s . Asterisks indicate a c t i v i t y s i g n i f i c a n t l y greater than the control (LSD, p<0.05). IPA = isopentenyl adenine. 134 chromatographic c h a r a c t e r i s t i c s common to the cytokinin-active components of the seaweed. Co-elution i n Gas Liquid Chromatography: Table 8 shows the results of an experiment designed to i d e n t i f y t e n t a t i v e l y the cytokinin-active constituents of seaweed c o l l e c t e d i n May, extracted and p u r i f i e d by the methods of Mukherji and Waring (1983) (Figure 3), and chromatographed on a column of XAD-2 according to Stafford et al.. (1984) (Section 3.2.2). Column eluates were dried and subjected to GLC analysis as per Section 2.4.3. Figures 24 shows examples of po s i t i v e co-elution with standard compounds. Figure 24A i s a chromatogram r e s u l t i n g from i n j e c t i o n of 1 u l of the putative zeatin-containing fr a c t i o n , and i l l u s t r a t e s peak enlargement when co-injected with s i m i l a r l y d e r i v i t i z e d zeatin. Si m i l a r l y Figure 24B shows co-elution of a compound i n the appropriate XAD-2 fr a c t i o n with isopentenyl adenine. It i s evident from these chromatograms that considerable contaminating material remained i n the p u r i f i e d extract. Thus two fractions contained compounds with appropriate extraction, XAD-2 elution, GLC retention times under two temperature programs, and bioassay responses (data not shown) for tentative i d e n t i f i c a t i o n as zeatin and isopentenyl adenine. 135 CO-ELUTION WITH IPA DHZ Z IPAR ZR EtOAc FRAC. Pl P2 Pl P2 Pl P2 Pl P2 P l P2 AF BR ID 0% - - - - - N 15% - - - - - N 50%A - - - - - Y 50%B - - - - - + N Y 100%A + + - - - - Y Y IPA 100%B + + + - - - - + N N n-BUT. FRAC. 0% - - - - - N 15% - - - + + N Y 50%A + + - + + - - - Y Y Z 50%B - - + + - - Y Y Z 100%A - - - + - - - N 100%B - - - - + - - - N TABLE 8 R e s u l t s of e x t r a c t i o n (Mukherji and Waring, 1983) chromatography on XAD-2 (Stoddart et al.,1984), b i o a s s a y (Miller,1956) and GLC (Kingman and Moore, 1982) of Macrocystis i n t e g r i f o l i a . XAD-2 e l u a t e s of the e t h y l a c e t a t e (EtOAc) and b u t a n o l i c (n-BUT.) e x t r a c t s were d r i e d ' b e f o r e b i o a s s a y and a n a l y s i s by GLC. C o - i n j e c t i o n of each c y t o k i n i n standard with each XAD-2 f r a c t i o n was performed, and peak enlargement at the a p p r o p r i a t e r e t e n t i o n time was sc o r e d p o s i t i v e . Lack of c o - e l u t i o n was scored n e g a t i v e . A b r e v i a t i o n s : IPA, i s o p e n t e n y l adenine; DHZ, d i h y d r o z e a t i n ; Z, z e a t i n ; IPAR, i s o p e n t e n y l adenosine; ZR, z e a t i n r i b o s i d e ; AF, a p p r o p r i a t e XAD-2 f r a c t i o n i n which the c o - e l u t i n g s t a n d a r d would normally appear; BR, bio a s s a y response; ID, t e n t a t i v e i d e n t i f i c a t i o n based on p o s i t i v e c o - e l u t i o n under two GLC temperature programs, l o c a t i o n i n a p p r o p r i a t e XAD-2 f r a c t i o n , and p o s i t i v e b i o a s s a y response; Y, yes; N, no; GLC, g a s - l i q u i d chromatography. GLC C o n d i t i o n s : S i l a n i z e d g l a s s column (2mm x 6') o f 3% SE-30 on GasChrome Q;N2 flow r a t e , 29 ml min. 1 ; FID temp. 350°C; i n j e c t o r temp, 250°C; oven temp., P l was 210°C 0-1 min., 22°C m i n . - 1 t o 265°C, 265°C 16 min., P2 was 185°C, 5°C m i n . - 1 to 2 6 5 ° C 136 Figure 24 Results of Co-chromatography of XAD-2 Eluates With Authentic Cytokin ins . 24A: Chromatagram of 100% methanolic eluate of an e thyl acetate f r a c t i o n from 50 gm of Af. integrifolia obtained by the methods of Mukherji and Waring (1983) . Dotted l ines indicate peak enlargement when co - in jec t ed with authentic isopentenyl adenine. The eluate was d r i e d for analys i s and d e r i v i t i z e d with BSTFA. 24B: Chromatogram of 50% (v/v) aqueous methanolic eluate of a butanol ic f r a c t i o n from 50 gm Af. integrifolia obtained by the methods of Mukherji and Waring (1983). Dotted l ines ind icate peak enlargement when co - in jec ted with authentic z e a t i n . The eluate was d r i e d for analys i s and d e r i v i t i z e d with BSTFA. XAD-2 Chromatography Condit ions: 2 cm x 15 cm glass column e luted with a stepwise gradient of 0%, 15%, 50%, and 100% (v/v) methanol (Stafford et al., 1984). GLC Condi t ions: S i l a n i z e d glass column (2mmx6') of 3% SE-30 on GasChrome Q; N 2 flow rate 29 ml m i n - 1 ;FID temp. 3 5 0 ° C ; i n j e c t o r temp. 2 5 0 ° C ; oven temp. 1 8 5 ° C , 2 min. , 30°C m i n . - 1 to 2 3 5 ° C , 2 3 5 ° C , 40 m i n . . 137 4. DISCUSSION In a l l three greenhouse experiments, plant responses to f o l i a r applications of sprays made from kelp were highly dependent on s o i l water p o t e n t i a l . This i s to be expected, since the physiological status of plants under such diverse environmental conditions would vary greatly. Some trends within each experiment, however, are of in t e r e s t . There were few s i g n i f i c a n t diferences among sprays made from kelp c o l l e c t e d throughout i t s l i f e cycle. Examination of the means for each variable revealed s o i l moisture-specific trends that were r e f l e c t e d i n the various s i g n i f i c a n t FC/D+W quadratic interactions. These are the result of d i f f e r i n g responses to the kelp sprays within each s o i l water pote n t i a l treatment. In dry s o i l , kelp c o l l e c t e d i n May made sprays that resulted i n generally higher mean y i e l d responses, although none were s i g n i f i c a n t in LSD comparisons within that water potential treatment. In f i e l d capacity s o i l , t h i s was true of spray made from kelp c o l l e c t e d i n July. Several variables, including leaf area and weight, stem weight and root weight were s i g n i f i c a n t l y greater than at least one other treatment i n LSD comparisons within that water regime. F i n a l l y i n wet s o i l , although again no s i g n i f i c a n t differences were evident among treatments, many y i e l d 138 variables were highest for plants treated with sprays made from kelp c o l l e c t e d i n May and January. Phytohormonal a c t i v i t y i n the kelp was highest i n May, and had declined i n July, reaching minimum levels i n September. Since in the stressed s o i l s , plant growth re s u l t i n g from sprays made from kelp harvested i n the spring resulted i n (non-significantly) higher y i e l d components, a relationship may exist between the high phytohormone-like a c t i v i t y of the May seaweed sample, and i t s effects when applied as a f o l i a r spray. Indeed a subsequent experiment demonstrated the ef f i c a c y of the cy t o k i n i n - l i k e f r a c t i o n of kelp c o l l e c t e d i n May when applied to plants grown under excess s o i l moisture conditions. However, for some y i e l d variables, plants sprayed with winter-harvested kelp preparations had (non-significant) higher production. Since phytohormone-like a c t i v i t y assessments were not performed on winter-collected kelp, the relationship of t h i s response to phytohormone-like a c t i v i t y i s unclear. Two papers have been published that addressed the question of seasonal phytohormone a c t i v i t y i n seaweed. Featonby-Smith and van Staden (1984a) reported that during the summer, cyt o k i n i n - l i k e substances with chromatographic mobility similar to those of free cytokinins predominated i n Ecklonia, while in winter, substances with the chromatographic behavior of cytokinin glycosides prevailed. 139 Total c y t o k i n i n - l i k e a c t i v i t y remained f a i r l y constant, perhaps s l i g h t l y higher i n spring, although the q u a l i t a t i v e changes were s i g n i f i c a n t . Another paper from the same laboratory (Mooney and van Staden, 1984) described changes i n the c y t o k i n i n - l i k e a c t i v i t y of non-reproductive fronds of Saroassum that show three yearly peaks in cytokinin a c t i v i t y , i n mid-summer, mid-winter, and spring. These peaks coincided with developmental aspects of seaweed growth. The results of t h e i r investigations indicated that large changes i n c y t o k i n i n - l i k e a c t i v i t y can occur from month to month, and that the q u a l i t a t i v e composition of c y t o k i n i n - l i k e substances can change likewise. The s l i g h t l y better performance of J u l y - c o l l e c t e d seaweed when applied to plants in f i e l d capacity s o i l could therefore be the result of q u a l i t a t i v e changes i n the phytohormonally-active components of the kelp, rather than through changes i n i t s gross content. Furthermore, the seasonal v a r i a t i o n i n other kelp constituents i s large (Whyte, 1975), and they could affect the absorption or e f f i c a c y of the active components of the seaweed spray. There was a point i n the evaluation of s h e l f - l i f e of stored kelp at which i t s e f f e c t on root and leaf weights was decreased on plants in dry and f i e l d capacity s o i l respectively. In addition, the height of plants i n wet s o i l was greatest for plants sprayed with un-aged kelp. 140 Furthermore, the depression i n root and leaf weights caused by f o l i a r application of l i q u e f i e d kelp stored for 118 days was not evident for kelp stored for longer durations. Transitory loss of a c t i v i t y may be explained by the appearance of i n h i b i t o r y degradation intermediates which depressed these variables. Since the kelp was macerated before storage, decompartmentalization of c e l l contents occurred, with r e s u l t i n g uncontrolled enzymatic and oxidative a c t i v i t y . The s o i l moisture and y i e l d component-specific nature of the response was unexplained by the available data. In wet s o i l , however, the e f f e c t on plant height was not recovered by longer storage of the kelp. This indicates breakdown of some kelp component that stimulated elongation, or a l t e r n a t i v e l y accumulation of a stable anti-elongation factor that i s e f f e c t i v e on flood-stressed plants. This e f f e c t was consistent with the stem elongation effects of g i b b e r e l l i n s , which are unstable (Hubick and Reid, 1984) and therefore l i k e l y to be degraded during storage; furthermore the supply of g i b b e r e l l i n s tends to be reduced under conditions of excess s o i l moisture (Reid and Railton, 1974). Since t h i s was the only persistent e f f e c t of storage, i t appears that storage of the kelp at room temperature for up to one year had l i t t l e e f f e c t . Thus, the growth promotive substances i n the kelp were stable. In the t h i r d greenhouse experiment, kelp c o l l e c t e d i n 141 May was fractionated by solvent p a r t i t i o n into four fractions by methods designed to separate various phytohormonal groups. Plant responses to spray applications were highly dependent upon s o i l water potential, as evidenced by the many s i g n i f i c a n t water potential by spray interactions. This was confirmed by LSD comparisons within s o i l water potential treatments. A trend was evident as water potential varied from flood, through f i e l d capacity to drought conditions. Comparison of the controls, sprayed with water, indicated that plants grown under dry conditions had lower y i e l d components than those under wet, conditions which i n turn were lower than those of plant's grown i n fi e l d - c a p a c i t y s o i l . Using y i e l d depression as a measure of the degree of stress, i t i s clear that plants grown in dry s o i l were more highly stressed than plants grown i n wet s o i l . For example leaf area and weight were reduced by about 20% on dry s o i l , but only by about 5% on wet s o i l . Height was reduced by 24% i n dry s o i l , and not at a l l in wet s o i l . The responses to drought conditions resembled those reported i n the l i t e r a t u r e (e.g. reduced height, reduced leaf area, reduced dry weights, and reduced moisture content of tissues) (Levitt, 1980; Salisbury and Ross, 1978; Burman and Painter, 1965). Control plant responses i n wet s o i l , however, only 142 partly resembled those described for flooded plants (entire root system submerged) in which root and shoot mass i s reduced, leaf senescence and abscision occurs, epinastic curvature i s evident, and flower abortion occurs (Jackson & Goss, 1978). In t h i s experiment some of these variables were reduced compared to plants i n f i e l d capacity s o i l s , others were not. For example, leaf area and weights were marginally reduced; stem weight and pod number were affected most, and root dry weights were unaffected. Y i e l d decreases due to root stresses can result from reduced photosynthetic rates that occur due to ABA-induced stomatal closure and alterations i n c e l l metabolism associated with senescence. ABA accumulation i n the shoots, of moisture-stressed plants (Walton, 1980; Jackson, 1984) can therefore contribute to y i e l d decreases. F o l i a r l y - a p p l i e d ABA can also i n h i b i t root growth (Wright, 1978). Cytokinins can oppose some ABA effects, as i n t h e i r a b i l i t y to delay or i n h i b i t senescence (Fletcher, 1969; Goodwin, 1978). Decock (197 9) reported that applied ABA reduced K and P content, while reducing Ca, Fe and starch l e v e l s . Application of BA and ABA together reduced these effects as well as reversing ABA-induced root i n h i b i t i o n . Cytokinin-like substances i n the seaweed may therefore have opposed these negative effects of ABA on plant growth. Since declines i n both cytokinin and g i b b e r e l l i n levels of Xylem sap of plants subjected to 143 flooding occurs (Burrows and Carr, 1969), phytohormone-like substances i n the seaweed may have ameliorated the e f f e c t s of these phytohormone d e f i c i t s i n the plants subjected to excess s o i l moisture. Railton and Reid (1974) demonstrated that the negative effects of flooding on tomato plants could be a l l e v i a t e d by f o l i a r applications of cytokinin. A mixture of cytokinin and g i b b e r e l l i n was even more e f f e c t i v e (Reid and Railton, 1974). In addition supplementing f o l i a r l y - a p p l i e d cytokinins with mineral nutrients can enhance t h e i r effects (Neumann and Neuden, 1983) thus other non-hormonal kelp constituents may also have contributed to the e f f e c t s . In general, however, according to Goodwin (1978) f o l i a r application of cytokinins i s i n e f f e c t i v e on whole plants. This contention i s rather too broad, since cases exist i n the l i t e r a t u r e that show p o s i t i v e responses to cytokinin application. Many, but not a l l , such p o s i t i v e responses to exogenous cytokinins involve stressed plants. Krishnamoorthy (1982) states that in the absence of adequate cytokinin, as in plants subjected to drought or s a l i n i t y , mobilization of amino acids and minerals from the leaves to other plant parts occurs. Benzioni et al. (1974) showed that k i n e t i n applied to s a l t - s t r e s s e d tobacco plants reduced necrosis, but decreased y i e l d . In f i e l d experiments, Crosby et al. (1978) got increases i n pod set by f o l i a r application of cytokinin 144 to soybeans, and Hassib et al.. (1971) obtained similar results, as well as an increase i n the number of seeds per pod. In a greenhouse experiment under conditions i d e n t i c a l to those described i n t h i s research, Olds (1985) showed that most b e n e f i c i a l results were obtained by treating bean plants grown under wet s o i l conditions with seaweed sprays made from Macrocystis i n t e g r i f o l i a . The results he obtained were simi l a r to those obtained with the c y t o k i n i n - l i k e fractions described in t h i s research. He also showed that changes i n y i e l d components and elemental content, as well as s o i l moisture and dose-response interactions were similar to those of BA-treated plants. This mimicry of seaweed spray effects by cytokinins has been demonstrated before on whole plants (e.g. Blunden and Wildgoose, 1977), as well as i s o l a t e d tissues. Finnie and van Staden (1985) mimiced the effects of seaweed preparations on root elongation and l a t e r a l root formation on in. v i t r o cultured tomato plants with very low concentrations of zeatin and i t s riboside. Wightman et a l . (1980) and Biddington and Dearman (1982) also showed cytokinin stimulation of root growth. These results support the suggestion that c y t o k i n i n - l i k e substances contributed to the e f f e c t s of the seaweed sprays, es p e c i a l l y in t h i e r stimulation of increased root/shoot r a t i o s . Plant responses to the various kelp fractions depended 145 on s o i l water p o t e n t i a l . The fr a c t i o n demonstrated to have cyt o k i n i n - l i k e a c t i v i t y due i n part to substances with physical properties similar to those of isopentenyl adenine and zeatin (Section 3.2.2), was e f f e c t i v e i n increasing most y i e l d variables under wet s o i l conditions. As discussed above, cytokinin levels in flooded plants are reduced, and replacement of t h i s d e f i c i t by exogenous cytokinins can ameliorate the effect of the d e f i c i t on plant growth. Increases i n general plant growth could also be the result of s p e c i f i c enhancement of root growth, with concommitant increases in nutrient interception and leading to growth increases (Widdowson et al.., 1973) . Interaction between factors described above could also occur. For example experiments have shown that the n u t r i t i o n a l status of the plant can affect i t s phytohormonal status, as well as i t s response to applied phytohormones (eg. Salama and Wareing, 1979, Marschner, 1988; Neumann, 1988). Thus an e f f e c t of applied growth substance on root growth, ion uptake capacity, or hydraulic conductivity could a l t e r endogenous phytohormone lev e l s , and thus the eff e c t of the applied chemical i t s e l f . In wet s o i l conditions application of the f r a c t i o n with c y t o k i n i n - l i k e a c t i v i t y raised the y i e l d of many variables to almost that of the control i n f i e l d capacity s o i l , yet for plants i n f i e l d capacity s o i l no eff e c t was observed. Thus, 146 i t i s possible that the e f f e c t of the f o l i a r l y applied c y t o k i n i n - l i k e substances i n t h i s f r a c t i o n was to raise stress-reduced cytokinin l e v e l s to a more productive l e v e l . Another p o s s i b i l i t y i s that application of t h i s treatment increased the endogenous cytokinin l e v e l s . Such an e f f e c t has been described by Featonby-Smith and van Staden (1984), who showed that application of the seaweed product Kelpak increased endogenous cy t o k i n i n - l i k e substance lev e l s i n f r u i t s , roots, leaves and stems of Phaseolus vulgaris by up to 10 f o l d . Application of l i q u i d f e r t i l i z e r alone also increased the c y t o k i n i n - l i k e substance content of the plant to a lesser degree. Application of both raised the t o t a l l e v e l i n the plant to an intermediate l e v e l , but the a c t i v i t y was concentrated i n the f r u i t s . This observation i s i n t e r e s t i n g i n the l i g h t of the increased pod dry weights produced from application of the f r a c t i o n with cytokinin-l i k e a c t i v i t y , since cytokinin accumulation i s usually associated with high nutrient sinks. They (Featonby-Smith and van Staden, 1984) suggest that more vigorous roots i n i t i a l l y stimulated by the f e r t i l i z e r and seaweed spray increased t h e i r nutrient u t i l i z a t i o n and cytokinin output, the net e f f e c t of which was increased above ground productivity. The response to a l l fractions i n dry s o i l was negative with respect to pod y i e l d . The complete unfractionated kelp 147 had no e f f e c t . Despite the fact that cytokinin delivery from the roots i s also reduced in drought conditions as described above, the cyt o k i n i n - l i k e f r a c t i o n of the kelp had a negative e f f e c t . Why then was growth not restored to the plant by replacing t h i s lost cytokinin, as i t may have been under wet s o i l conditions? Evidently the constraint on growth was imposed by some factor not supplied or induced by the kelp treatments. Plant productivity was apparently maximal under those conditions, and perturbations reduced y i e l d even further. It i s evident from the water-sprayed controls that the stress l e v e l was higher for plants i n dry s o i l than i n wet s o i l . The applied c y t o k i n i n - l i k e substances of al g a l o r i g i n may have been i n s u f f i c i e n t to restore growth under these extreme stress conditions. Also, the n u t r i t i o n a l status and water balance was d i f f e r e n t under water excess and d e f i c i t conditions. Therefore, t h e i r phytohormonal balances were also d i s s i m i l a r and they would respond d i f f e r e n t l y to applied phytohormone-like substances. Furthermore, f o l i a r absorbtion of applied chemicals in general depends to some degree on leaf surface c h a r a c t e r i s t i c s . Absorbtion of some components of the spray may thus have been altered by changes in leaf structure r e s u l t i n g from drought stress. These adaptations, such as reduced leaf area, reduced stomatal area, and increased c u t i c u l a r resistance, are designed to reduce water 148 loss (Salisbury and Ross, 1978, Lev i t t , 1980), but would also reduce f o l i a r absorbtion. The negative e f f e c t on call u s growth of increasing amounts of extracted kelp indicated that there may be an in h i b i t o r y substance i n the seaweed that reduced growth. The presence of i n h i b i t o r s in seaweed extracts i s described in the l i t e r a t u r e (Jennings, 1969), and growth of i n v i t r o cultured tomato roots seem to show i n h i b i t i o n to some Rf zones of chromatographed seaweed extract (Finnie and van Staden, 1985) . The f r a c t i o n putatively containing some cytokinins primarily of tRNA o r i g i n (Sembner et al.., 1980), esters of IAA and some glucosyl esters of some gi b b e r e l l i n s (Rademacher and Graebe, 1984), also demonstrated growth enhancing a c t i v i t y , but only in f i e l d capacity s o i l s . Variables affected included leaf area ans weight, and number of pods produced. As with the performance of the f r a c t i o n with c y t o k i n i n - l i k e a c t i v i t y on plants in wet s o i l , some of the responses were p a r a l l e l l e d , but to a lesser degree, by non-s i g n i f i c a n t increases caused by the unfractionated kelp spray. The effects of these more obscure phytohormones when applied exogenously i s not described i n the l i t e r a t u r e , nor has t h e i r occurrence in algae been confirmed. Thus, i t i s d i f f i c u l t to speculate on the o r i g i n of these observed growth e f f e c t s . It i s possible that some of these compounds are 149 p r e s e n t i n a l g a e as a r e s u l t o f m e t a b o l i s m o f IAA known t o be p r e s e n t ( e g . J a c o b s e t a l . , 1 9 8 5 ) . H i g h e r p l a n t s h a v e t h e c a p a c i t y t o c o n v e r t s u c h c o n j u g a t e s t o IAA (Sembner et. a l . f 1 9 8 0 ) / a n d t h u s t h e compounds t h e m s e l v e s u s u a l l y h a v e some b i o a s s a y a c t i v i t y . A u x i n b i o a s s a y o f t h i s f r a c t i o n , h o w e v e r , showed no a c t i v i t y i n t h e A v e n a e l o n g a t i o n b i o a s s a y ( d a t a n o t s h o w n ) , h o w e v e r t h e e x t r a c t was n o t c h r o m a t o g r a p h e d p r i o r t o b i o a s s a y a n d b i o l o g i c a l a c t i v i t y may t h e r e f o r e h a v e b e e n masked. F u r t h e r m o r e , e s t e r s o f IAA a r e e a s i l y h y d r o l y s e d t o IAA d u r i n g e x t r a c t i o n (Goodwin, 1978) a n d t h e I A A t h u s f o r m e d w o u l d c o m p r i s e p a r t o f a n o t h e r f r a c t i o n w h i c h h a d IAA b i o a s s a y a c t i v i t y . The f r a c t i o n d e m o n s t r a t e d t o h a v e IAA a c t i v i t y m a r g i n a l l y a f f e c t e d l e a f w e i g h t i n f i e l d c a p a c i t y s o i l , a n d s u b s t a n t i a l l y i n c r e a s e d t h e number o f p o d s . T h i s s i m i l a r i t y i n r e s p o n s e may i n d e e d be t h e r e s u l t o f I A A - l i k e compounds p r e s e n t i n b o t h f r a c t i o n s . E x o g e n o u s a p p l i c a t i o n o f compounds w i t h a u x i n a c t i v i t y h a s b e e n u s e d i n a g r i c u l t u r e f o r many y e a r s ( K r i s h n a m o o r t h y , 1 9 8 2 ) . E l B a l t a g y e t a l . (1979, c i t e d i n K e l l e r a n d B e l l u c c i , 1983) d e m o n s t r a t e d t h a t i n d o l e b u t y r i c a c i d a p p l i c a t i o n t o f a b a b e a n s i n c r e a s e d t h e number o f f l o w e r s , a nd E l A n t a b l y (1976) showed a d e c r e a s e i n a u x i n c o n t e n t o f s o y b e a n p o d s j u s t p r i o r t o p o d d r o p . A l s o , W i t t w e r (1983) s t a t e s t h a t a p p l i e d a u x i n s h a v e b e e n e f f e c t i v e i n i n c r e a s i n g f r u i t s e t i n s n a p b e a n s . P e r h a p s t h e a u x i n 150 a c t i v i t y of these fractions resulted i n similar e f f e c t s , thus increasing pod number. These experiments have demonstrated that f o l i a r sprays made from M. i n t e g r i f o l i a have the potential for use i n improving bean production under some conditions. The e f f i c a c y of some fractions i n the greenhouse, and of the complete spray in the f i e l d and i n the greenhouse (Olds, 1985; Temple and Bomke, 1988; Temple et a l . , 1989) v e r i f i e s t h i s p o t e n t i a l . Since the e f f e c t was associated with root stress, and some negative effects were observed i n drought conditions, more work must be done to define the conditions under which the f o l i a r spray should be used. Although the mechanism by which growth promotion occurred remains undefined, the work described above lends circumstantial evidence to the hypothesis that under some conditions the phytohormone-like substance content of the kelp contributes to i t s e f f e c t s . Further investigation i s therefore warranted. Since d i f f e r e n t fractions of kelp affected growth d i f f e r e n t l y , the interaction among these fractions deserves consideration. A companion experiment to the fractionation experiment described above, i n which of recombined fractions are tested would shed l i g h t on t h i s . Exclusion of single fractions, one by one, with recombination of the remaining 151 ones would allow assessment of fra c t i o n i n t e r a c t i o n . Further research might also include s p e c i f i c removal of phytohormones from the unfractionated kelp by passage through immunoaffinity columns (eg. Senapathy and Jacob, 1981). Loss of a c t i v i t y of the column eluates would then v e r i f y a contribution by that s p e c i f i c phytohormone to the responses. Description by more rigorous physico-chemical means (eg Hubick and Reid, 1984), and a mimicry of the kelp spray effects by authentic phytohormone combinations that match those i n the spray would also strengthen the hypothesis. 152 5. SUMMARY/CONCLUSIONS Summary 1) Responses of Phaseolus vulgaris to f o l i a r application of the kelp Macrocystis i n t e g r i f o l i a were dependent on s o i l moisture regime. 2) Responses to kelp c o l l e c t e d throughout i t s l i f e cycle indicated that sprays made from kelp c o l l e c t e d i n July had greater effects on some bean plant y i e l d variables i n f i e l d capacity s o i l s than kelp c o l l e c t e d in other months. 3) Responses to kelp stored for various durations at room temperature indicated a transient difference i n effects on leaf and root weights of plants grown i n dry and f i e l d capacity s o i l that was not evident when the kelp was stored for longer or shorter duration. In wet s o i l s , bean plants sprayed with aged l i q u e f i e d kelp were shorter than plants sprayed with un-aged l i q u e f i e d kelp. 4) Relative gross c y t o k i n i n - l i k e and auxin-like bioassay a c t i v i t y detected was greatest i n kelp samples co l l e c t e d i n May and decreased in samples co l l e c t e d July through 153 September: changes i n levels appeared not to be d i r e c t l y related to e f f i c a c y . 5) A butanolic extract of kelp harvested i n May caused s i g n i f i c a n t increases in many y i e l d variables of plants grown in excess soil-moisture conditions. The f r a c t i o n had no effe c t when plants were held at f i e l d capacity, and, l i k e a l l other treatments except the unfractionated kelp spray, decreased pod y i e l d under dry s o i l conditions. 6) This extract was further analysed and shown to exhibit c y t o k i n i n - l i k e a c t i v i t y , some of which had the chromatographic and b i o l o g i c a l c h a r a c t e r i s t i c s of isopentenyl adenine and zeatin. 7) A f r a c t i o n with auxin and g i b b e r e l l i n - l i k e a c t i v i t y caused some b e n e f i c i a l y i e l d e f f e c ts on plants grown i n f i e l d capacity s o i l , as did another fr a c t i o n with undefined phytohormone-like a c t i v i t y . 8) The depression in pod y i e l d under dry s o i l conditions caused by a l l fractions, as well as decreased c y t o k i n i n - l i k e a c t i v i t y per gram of tissue with increased weight of kelp assayed, indicated the presence of i n h i b i t o r s i n the kelp. 154 9) The y i e l d increases caused by the fr a c t i o n containing c y t o k i n i n - l i k e substances may be related to the amelioration of phytohormonal d e f i c i t s caused by root stress. 10) The po s i t i v e effects of another f r a c t i o n (Fraction II) on plants grown i n s o i l at f i e l d capacity may be due to some phytohormonal derivatives, and requires further investigation. 11) Further investigation of the use of al g a l f o l i a r sprays in crop production i s warranted to t r y to define the environmental and developmental conditions that optimize t h e i r e f f e c t s . 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