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Effects of peat and sawdust mulches and their leachates on the growth and certain metabolic reactions… Osborne, James Robert 1961

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THE EFFECTS OF PEAT AND SAWDUST MULCHES AND THEIR LEACHATES ON THE GROWTH AND CERTAIN METABOLIC REACTIONS OF THE HIGHBUSH BLUEBERRY ( V a c c i n i u m corymbosum L . v a r C o v i l l e )  BY  JAMES ROBERT OSBORNE, B.S.A.  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE  We a c c e p t t h i s t h e s i s a s conforming t o t h e s t a n d a r d requirement from c a n d i d a t e s f o r t h e Degree o f Master o f S c i e n c e i n Agriculture•  Members o f t h e D i v i s i o n o f P l a n t  Science  THE UNIVERSITY OF BRITISH COLUMBIA April, 1961  In presenting t h i s t h e s i s i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study.  I further agree that per-  mission f o r extensive copying of t h i s t h e s i s f o r scholarly purposes may  be granted by the Head of my Department or by h i s represen-  tatives.  I t i s understood that copying or publication of t h i s  thesis f o r f i n a n c i a l gain s h a l l not be allowed without my permission.  Department of Horticulture The University of B r i t i s h Columbia, Vancouver 8 , Canada. Date A p r i l ,  1961.  written  ABSTRACT Greenhouse, f i e l d and laboratory experiments were conducted t o a s c e r t a i n some growth and metabolic responses of the C o v i l l e blueberry v a r i e t y , t o sawdust and peat mulches, and the leachates o f these mulches. From the greenhouse experiments i t would appear that i n some manner the mulches i n question increased the metabolic a c t i v i t i e s of the p l a n t s .  The increased a c t i v i t y was r e f l e c t e d  by increased growth o f the mulched p l a n t s , a higher ash content and a higher t o t a l nitrogen content o f the leaves. A greater concentration o f free amino acids i n the unmulched plants than i n the mulched ones;  ocourred  This indicates  that the nitrogen metabolic a c t i v i t i e s were hastened by the mulching.  AOKMOWLEDGrEMEKTB  The w r i t e r wishes to express h i s thanks t o Dr. G, H. Harris, Professor of H o r t i c u l t u r e , whose t e c h n i c a l advice, time spent, and information offered was of inestimable value i n the preparation of t h i s t h e s i s .  TABLE OF CONTENTS  Introduction  1  Review of Literature  3  Mater ialB and Methods F i e l d Experiments Greenhouse Experiments Results o f Experiments Table I . ., The E f f e c t o f Treatments on the pH of the percolates after passing through the growing medium Table I I The effect o f peat and sawdust mulches, steam-treated and untreated leachates on the accumulative growth of blueberry plants (length o f shoot measured i n c e n t i metres)  17 17 13 23  23  24  Table I I I S t a t i s t i c a l Analysis of Increase i n Shoot Growth ( i n Centimetres)  25  Table I I I A Treatments and accumulative mean shoot growth arranged i n Descending order  26  Table 17 The effect of sawdust and peat mulches on the average moisture, ash and t o t a l nitrogen content of the leaves of blueberries, (figures represent averages of 12 determinations.)  27  Table Y The Rf values and ninhydrin colour o f amino-acids found i n the leaves o f unmulched, sawdust mulched and peat mulched plants compared with standard values of certain ami no-a c ids  28  Figure I Photograph of a t y p i c a l chromatogram comparing aminoaclds i n samples from leaves of the unmulched and peat mulched plants  29  Table VI A r e l a t i v e evaluation of the nitrogen f r a c t i o n found i n the leaves of unmulched, peat mulched and sawdust mulched plants i n the form o f amino-acids  30  Page Discussion of Results  31  Conclusions  3  Sunmjaryu.  37  Literature c i t e d ..;  39  Appendix %  43  Appendix I I  44  /•.rsAppendix I I I  45  6  1 IMTRODTJCTION  Experiments i n blueberry culture were i n i t i a t e d by F.V. C o v i U e i n 1906. He selected and crossed w i l d species of high and low bush form and from 1921 t o 1939 produced 18 named varieties.  In 1949 these 18 v a r i e t i e s constituted the entire  commercial acreage of highbush blueberries i n America with the exception o f Rubel, a v a r i e t y selected from the w i l d by Miss E.C. White o f Whitesborg, New Jersey. At h i s death i n 1937 Dr» O o v i l l e l e f t many thousands of seedlings which have given r i s e t o further superior v a r i e t i e s . Today there i s a t o t a l of about 4,000 acres o f the c u l t i v a t e d highbush blueberry i n the United States.  In Canada  the industry i s small but i s rapidly expanding i n the Maritimes, Ontario, and B r i t i s h Columbia;  There are now some 800 acres of  blueberries planted i n the lower mainland coastal area of B r i t i s h Columbia. Because o f the a v a i l a b i l i t y of large acreages i n B r i t i s h Columbia p o t e n t i a l l y suitable for blueberry production, together with a b r i s k and increasing demand f o r the f r u i t ,  i t was f e l t that  a contribution i n the f i e l d of blueberry n u t r i t i o n would be of value i n f o s t e r i n g t h i s neophyte B.C. Industry. In c e r t a i n blueberry growing areas the cultivated blueberry appears t o s u f f e r from various n u t r i t i o n a l disorders.  Poor  growth has been attributed to such f a c t o r s as magnesium, iron and  2 manganese d e f i c i e n c i e s , unfavourable pH and nitrogen r e l a t i o n ships.  A number o f remedies have been t r i e d with varying degrees  of success.  On mineral s o i l s the use o f mulching materials has  i n c e r t a i n cases, stimulated growth and improved the health and vigour o f the p l a n t s . It was decided to investigate the effect of peat and sawdust mulches on the growth and vigour of the blueberry.  Further-  more, as an i n d i c a t i o n o f the n u t r i t i o n a l status o f the plants i t was considered adviseable t o a s c e r t a i n the effect o f the mulches on the moisture content, t o t a l mineral content (ash) and t o t a l nitrogen content o f the leaves.  I t was also f e l t that a study of  the free amino-acids i n the leaves would be h e l p f u l i n understanding the metabolic a c t i v i t y of the p l a n t .  3  REVIEW OF LITERATURE Some E f f e c t s of Mulches on Blueberries, A comparison of the effect of sawdust and straw mulches with a buckwheat cover crop and clean c u l t i v a t i o n was made by Shutak and Christopher (46) i n Hew Jersey using the Pioneer variety of blueberry.  They found that consistently higher y i e l d s  were produced by bushes mulched with sawdust as contrasted t o clean c u l t i v a t e d p l o t s which produced the lowest y i e l d s .  The sawdust  mulch had very l i t t l e effect on the pH of the s o i l but s o i l temperatures were lower i n spring and summer and higher i n the f a l l under the mulch. the  A larger and mare fibrous root system developed under  sawdust mulch than with the other treatments.  These workers  also reported that weed control was greatly s i m p l i f i e d by the use of the sawdust mulches. Chandler (18) reported that under conditions i n Maine, the  s o i l mulched with sawdust under blueberry plantings, retained  more moisture and maintained lover temperatures than i n unmulched plantings.  The mulch increased growth of plants i n clay and loam  s o i l s , but on the other hand reduced growth o f plants growing i n sandy s o i l s . In Georgia, Savage (45) found that looser forms of mulches such as rye, straw, and oak leaves were better than clean c u l t i v a t i o n , but not as good as sawdust. Kramer (33) i n Maryland reported increased y i e l d s of both high and dryland blueberries were obtained with various mulches  4  although mulching decreased s u r v i v a l of the dryland blueberries. Mulching with sawdust i n Connecticut gave greater growth and better y i e l d s than e i t h e r clean c u l t i v a t i o n or hay mulch according to Griggs and R o l l i n s (26).  The d i f f e r e n t types of s o i l manage-  ment however did not a f f e c t the moisture content or the ascorbic a c i d content of the berries* Several workers (2, 23, 47, 48) have reported that f r e s h sawdust depressed a v a i l a b l e s o i l nitrogen, but that rotted sawdust was an excellent s o i l a d d i t i o n . Johnston (29) found that sawdust alone used as a surface mulch depressed n i t r a t e s i n the s o i l s l i g h t l y f o r the f i r s t year and further depressed i t for a second year but when the sawdust was incorporated with the s o i l the nitrates were depressed f o r 18 months a f t e r which time they accumulated. According t o Turk (47), i n Michigan an average sawdust contains 4 l b s . o f nitrogen, 2 l b s . phosphorous pentoxide and 4 l b s . of potash per ton of dry material.  This i s about one-third the amount  of nutrients i n the same weight of wheat straw. Comparing the e f f e c t s of clean c u l t i v a t i o n , sawdust mulch, oak leaf mulch, and rye straw mulch on blueberry plants, Savage and Darrow (45) found the plants from mulched p l o t s were superior to those from clean c u l t i v a t e d p l o t s and that sawdust was superior to either oak leaves or rye straw.  On the basis of plants surviving at the  end of the t h i r d year the sawdust mulch was superior to rye straw  5  and oak l e a f mulch.  The plants In the clean c u l t i v a t e d p l o t s  were so small that, ccmnercially t h e i r productive  capacity was  regarded as valueless* Anderson (1) reported that the a d d i t i o n of straw and c e l l u l o s e depressed available? nitrogen i n the s o i l .  Turk and  Partridge (47) found that n i t r i f i c a t i o n was slower i n mulched than In unmulched s o i l s , but due t o leaching losses i n the unmulched p l o t s , the t o t a l n i t r a t e accumulation was the same i n a l l p l o t s at the end of his experiment. Shutak (46) found that sawdust mulches increased the water holding capacity o f the s o i l i n blueberry p l o t s but that the increase was not as great as when straw mulch was used. Turk (47) reported that the effect of sawdust i s l a r g e l y physical, r e s u l t i n g i n a better water holding capacity, decreased s o i l losses due t o erosion and the prevention of a hard crust on the s o i l surface l i m i t i n g the c a p i l l a r y movements of water. Johnston (30) found that a surface mulch of sawdust i n creased the s o i l moisture and that the s o i l temperature was more uniform under the mulch. In order to eliminate any e f f e c t s of the increased moisture holding capacity of the s o i l due to the mulch Harris (28) provided uniform moisture i n a l l p l o t s with i r r i g a t i o n and found that a sawdust mulch markedly increased the y i e l d s of strawberries i n a sandy s o i l compared t o that of unmulched plants*  6  Blueberry N u t r i t i o n , Doehlert and Shive (22) working with both sand cultures and f i e l d p l o t experiments to determine the n u t r i t i o n a l needs of the c u l t i v a t e d blueberry, (Vaccinium  corymbosum), reported that the  best nutrient solutions for the blueberry were low i n phosphorus and high i n nitrogen.  N i t r a t e nitrogen appeared to be of greater value  than ammonium nitrogen f o r t h i s p a r t i c u l a r species. was  Good agreement  obtained between the r e s u l t s found i n the sand cultures and those  found i n the f i e l d p l o t s .  The blueberry was  to deficiency o f boron and manganese. aamonium nitrogen was  shown t o be s e n s i t i v e  Cain (16) reported that  superior to n i t r a t e nitrogen f o r blueberries  i n contrast to the r e s u l t s found by Doehlert and Shive (22).  Plants  which received n i t r a t e nitrogen showed i r o n deficiency symptoms more r e a d i l y than those which received ammonium n i t r a t e although the iron content o f the leaves i n both cases was ammonium nitrogen was  i n some way  similar.  He concluded that  associated with iron n u t r i t i o n .  Perlmutter and Darrow (40) under the conditions of t h e i r experiment, did not obtain any benefit from applications of various forms of nitrogen f e r t i l i z e r s . They a l s o concluded that s o i l pH may  not necessarily be a  direct c o n t r o l l i n g factor i n i r o n absorption because plants growing on s o i l s of r e l a t i v e l y high pH often contained as much iron i n t h e i r f o l i a g e as those growing at a lower pH. In a study of a c h l o r o t i c condition of blueberry leaves, Mikkelsen and Loth (37) found that green leaves contained 350  ppm.  7  of soluble magnesium as compared with 30 ppm  i n the c h l b r o t i c  leaves. McHargue (36) reported that manganese i s concerned i n nitrogen a s s i m i l a t i o n and i n the synthesis o f proteins and that i t functions as a catalyst i n plant metabolism and also functions with i r o n i n the synthesis of chlorophyll. Importance of pH i n Blueberry  Culture.  A number of investigators (8,19,27, and 30) have stressed the importance of s o i l a c i d i t y for blueberry growth, suggesting range of pH of 4.0  to 5.2  be expected to survive.  a  above or below which blueberries may B o i l e r (13), however claimed that  not  soil  structure, aeration, organic matter and water are the most important factors i n blueberry culture and that i f these conditions are s a t i s factory, plantings could be maintained over a wide pH range. Cain (17) concurred with B o i l e r and reported plantings on s o i l s above pH 6.0  successful  i n the f i e l d and at pH 6.5  solutions when an adequate nutrient supply was maintained. sidered that s o i l pH was  i n nutrient He con-  not n e c e s s a r i l y a d i r e c t c o n t r o l l i n g factor  i n i r o n absorption because plants growing on s o i l s of r e l a t i v e l y high pH often contained as much iron in t h e i r f o l i a g e as those growing at a lower  one. Spraying  ed by Blasberg  the f o l i a g e w i t h soluble i r o n s a l t s was recommend-  (10) to correct c h l o r o s i s associated with high  pH  s o i l s , but only as a temporary corrective measure because the chloros i s usually reappeared i n new  growth made a f t e r the spray a p p l i c a t i o n .  8  Bailey and Everson (6) corrected chlorosis with s o i l a p p l i c a tions o f ammonium sulphate or aluminum sulphate.  T hey found  soluble i r o n available i n the s o i l as a result of t h e i r t r e a t ments. Beckwith (7) suggested that ammonium sulphate gave better growth of blueberry plants i n s o i l s above pH 5.5  and  Doehlert (21) recommended i t s use i n connection with lime on commercial blueberry plantings. Johnston and Ware (31) performed experiments using sawdust as mulch; and they reported that differences i n pH of s o i l s mulched with sawdust and not mulched were small. Manganese i n Blueberry N u t r i t i o n . McHargue (36) reported that manganese i s concerned i n nitrogen a s s i m i l a t i o n and i n the synthesis of proteins; and that i t functions as a catalyst i n plant metabolism and a l s o functions with iron i n the synthesis of chlorophyll.  He considered also thaft-.  since low manganese a f f e c t s the production of dry matter, the i n dications are that i t has some effect upon carbon a s s i m i l a t i o n . T y p i c a l symptoms of manganese deficiency i n plants are:the terminal bud remains a l i v e and leaves do not w i l t ; chlorosis i s present with spots o f necrotic tissue scattered over the leaf; the small veins tend t o remain green giving the leaves a checkered or r e t i c u l a t e d e f f e c t (36). On the other hand manganese excess i s t y p i f i e d i n i t i a l l y by a yellowing of the leaves accompanied by s l i g h t upward c u r l i n g of the upper leaves.  The chlorosis continues u n t i l the newer  9 leaves are almost white.  The leaves then c u r l down and the mid-  r i b s often darken and break down.  Large necrotic areas f i n a l l y  develop i n the c h l o r o t i c leaves and t h i s i s accompanied by the breakdown and death of the stem (36). Magnesium i n Blueberry N u t r i t i o n . Mikkelsen and Loth (37) established the fact that the soluble magnesium contents of green and c h l o r o t i c leaves were different.  Normal green leaves contained 350 ppm o f magnesium as  compared with 30 ppm. i n the c h l o r o t i c leaves.  S o i l samples c o l l e c t -  ed near affeoted p l a n t s contained only a trace of a v a i l a b l e magnesium. Kramer and Schrader (33) studied the e f f e c t s o f mineral d e f i c i e n c i e s on the growth of the Cabot blueberry i n sand c u l t u r e s . They described magnesium deficiency as a marginal l e a f c h l o r o s i s of the basal leaves extending i n t r a - v e i n a l l y with green midrib.  The  l a t e stages o f t h i s deficiency a r e characterized by the chlorotio areas becoming red.  This characterization of magnesium deficiency  has been observed i n the f i e l d on the Jersey, June, Cabot, and Stanley v a r i e t i e s i n New Jersey, except that necrosis has been r a r e . Very few data are available on the rates of s o i l a p p l i c a t i o n o f magnesic f e r t i l i z e r s necessary t o correct magnesium deficiency.  Mikkelsen and Doehlert  (37) reported that magnesium  deficiency i n blueberries was corrected by applying magnesic materials to the s o i l .  F a l l applications of 70 to 300 pounds of MgO per acre  supplied as Epsom s a l t s and hydrated dolomitic lime, r e s p e c t i v e l y , corrected the deficiency during the following season.  Magnesic f e r -  t i l i z a t i o n increased the magnesium content of the leaves and elevated  10 the m i l l i e q u i v a l e n t cation composition o f them, while phosphorus, i r o n and manganese composition was r e l a t i v e l y unaffected. The foregoing indicates that there i s disagreement i n the value of mulches used i n blueberry culture and that the e v i d ence c o n f l i c t s as to the various causes of poor growth. The following i s a review of chromatography methods used f o r the detection of free amino-acids. Qualitative Paper P a r t i t i o n  Chromatography.  Consden, Gordon, and Martin (20) have shown that good separation ot amino-acids can be obtained on f i l t e r paper by allowing a suitable solvent, which has previously been saturated with water to flow over the paper i n a closed container, the a i r i n which was saturated with the vapours of water and the solvent. F i l t e r paper contains 20-25 per cent water under these cond i t i o n s , and separation depends upon the differences i n p a r t i t i o n coe f f i c i e n t of the amino-acids between the stationary water phase and the moving solvent.  I t has further been shown that f o r any i n d i v i d -  ual amino-acid the value: Rf  =  distance moved by the amino-acid distance moved by t h e advancing front of l i q u i d  i s d i r e c t l y r e l a t e d to the p a r t i t i o n c o e f f i c i e n t , true absorption by the cellulose playing l i t t l e part. Qualitative Analysis of Amino-acids. In an experiment on the q u a l i t a t i v e micro-analytical technique f o r amino-acids, Consden, Gordon and Martin (20) reported on: 1. Rate of d i f f u s i o n of Amino-acids i n various solvents.  11  A number of solvents have been t r i e d and abandoned. Of these e t h y l acetate, methylethyl ketone, a n i l i n e , cyclohexanol, cyclohexane, quinoline and a l i g h t a n i l i n e f r a c t i o n b.p. 174-180 were unsatisfactory as the amino-acids ran too slowly, whereas with methyl acetate, acetone, sec-butanol, pyridine, the p i c o l i n e s and the l u t i d i n e s , the bands of amino-acids either moved too fast or were unduly broadened.  Hydroxy-amino-acids move more slowly  than the corresponding amino-acids i n phenol, but i n c o l l i d i n e the rates are s i m i l a r . Ammonia s e l e c t i v e l y slows the movement of aspartic and glutamic acids and hastens that of basic amino-acids.  A c i d has the  reverse e f f e c t . 2.  Additions of Acids Bases and S a l t Hydrolysates, or mixtures  in which the r a t i o o f soluble inorganic s a l t s of amino-acid were high, gave unsatisfactory chromatograms.  The s a l t absorbed water  from the atmosphere and the solvent and caused l o c a l water-logging of the paper.  The amino-acids were not readily washed from these  regions and the r e s u l t i n g bands were grossly d i s t o r t e d .  This s a l t  effect was eliminated by impregnating the paper with s a l t and using the solvent and atmosphere equilibrated with saturated s a l t s o l u t i o n instead of with water. Micro Estimation of Amino-nitrogen. Pope and Stevens (43) described a method f o r determining amino and peptide nitrogen.  Woiwood (50) successfully adapted t h i s  method to the determination of micro amounts ( 1 - 2 5 amino-nitrogem.  ugm) of  eC -  The e s s e n t i a l points of the method were that a f t e r  12 the amino-acid was allowed to react with copper phosphate, the s o l uble copper complex i n the f i l t r a t e was decomposed by means of sodium-dithio-earbamate, the resultant c h a r a c t e r i s t i c golden colour was extracted i n amyl alcohol and the amount o f copper determined absorptiometrically. of  fi-  The t h e o r e t i c a l value of 0.44  f o r the r a t i o  amino-nitrogen to copper i n the complex AgCu was not ob-  tained under the experimental conditions used, i t was essential to construct a standard curve f o r each i n d i v i d u a l amino-acid. Such curves were reproducible, and over t h e i r l i n e a r port i o n (10 - 25 ugm,  «C- amino-nitrogen) the amino-acids methionine,  hydroxyproline, aspartic a c i d , leucine, isoleucine, h i s t i d i n e , phenylalanine, valine, glutamic acid, tryptophane and tyrosine gave r a t i o s l y i n g between 0.50 and 0.55; alanine, glycine, threonine, serine, cystine and cysteine r a t i o s between 0.55 and 0.80; and l y s i n e , arginine and glucosamine r a t i o s between 12.5 and  1.6.  Quantitative Paper Chromatography. Quantitative A p p l i c a t i o n o f Paper Chromatography to Amino A c i d s. One of the two major problems i n developing a method f o r quantitative determination o f amino-acids by paper p a r t i t i o n chromatography i s the measurement of the amino-acids a f t e r they have been chromatographed.  A number of procedures have been proposed which  include colorimetric and polarographic methods as w e l l as the use of photoelectric densitometer. Poison et a l (42) were among the f i r s t to report a  13  quantitative procedure f o r determining amino-acids a f t e r separat i o n by paper p a r t i t i o n chromatography.  The technique included  the development of a colour with amino compounds and ninhydrin on the paper, the extraction of the colour with acetone and the measurement of the colour. Aside from the f a c t that the acetone i s a poor solvent for the coloured product, these authors apparentl y found other disadvantages to t h i s procedure because i t was  ab-  andoned f o r one i n which comparisons were made with standard amounts of amino-acids chromatographed simultaneously with unknowns. This technique has the disadvantage that i t requires a large number of standards with each sample, or set of samples, and i t depends on a subjective test which can only be approximately quantitative. Several subsequent methods have u t i l i z e d one-directional chromatography, formed a colour with ninhydrin, and measured the colour by the density or s i z e of coloured areas.  Fisher et a l (24)  measured the area of the spot formed by the ninhydrin r e a c t i o n , and found that the s i z e was proportional to the logarithm of the concentration of the amino-acid.  The concentration of an amino-acid  i n a sample can then be determined by comparison with standards run simultaneously.  One technique they used was a photographic method  for determining a spot s i z e . B u l l et a l (15) determined the l i g h t transmission of the coloured compound on the paper at d i f f e r e n t points along the spot  14  and i n the d i r e c t i o n of phenol movement. By integrating the area under the curve r e l a t i n g colour density and distance, an estimate of quantity was made.  Block (12) used a s i m i l a r method  except that the amino-acids were f i r s t separated into a c i d i c , basic, and neutral amino-acids chromatography.  on ion exchange resins before  In the case of two-directional chromatograms,  Block (12) determined the quantity of amino-acid by multiplying the maximum colour density of the area of the spot. Block (12) has developed t h i s method further i n order to obtain the percentage composition of an amino-acid mixture. The method i s more adapted to r e l a t i v e rather than absolute values. The maximum i n t e n s i t y of a given spot was found to give an estimate of the quantity of each amino-acid,  The maximum colour density of  the spot obtained f o r each amino-acid was determined at the same molar concentration. The r a t i o of the colour density f o r a p a r t i c u l a r aminoa c i d to the sum of the colour densities of a l l the amino-acids  con-  s t i t u t e a "standard colour r a t i o f o r the amino-acid i n question". An "experimental colour r a t i o ? ' f o r each amino-acid i s determined i n the same way  i n a sample of the mixture to be analysed.  The "experimental colour r a t i o " divided by the "standard colour r a t i o " gives the molar r a t i o of each amino-acid i n the mixture.  Analysis of casein by the empirical method agreed c l o s e l y  with published values although a large number of chromatograms (11)  15  were necessary to achieve t h i s , Rockland and Dunn (44) used a densitometrie method f o r glycine and alanine.  The amino-acids were separated on one dim-  ensional chromatograms and the colour produced by ninhydrin. By measuring the density of ah area larger than the biggest spot i t was possible to measure the density due to amino-acid by d i f f e r e n c e . In the analysis of amino-acids i n urine by chromatography, Berry and Cain (9) u t i l i z e d v i s u a l comparisons o f spots.  Owing,  however, to the effect op. the chromatography o f water-soluble materials i n the urine, the standards with which comparisons were to be made were prepared by adding known amounts of amino-acid urine.  Although the authors claim 10 to 15 percent accuracy o f  t h e i r methods, the recovery o f added a c i d varied over as wide a range as 63 to 160 percent, A novel method somewhat analogous to that of B u l l et a l (15) was introduced by Keston et a l (32),  The amino-acids were  made to react with a compound containing radio-active iodine and the r e s u l t i n g compounds were chromatographed i n n-pentanol saturated with 2N ammonium hydroxide.  The r a d i o a c t i v i t y was measured i n the  d i r e c t i o n of solvent movement, and s p e c i f i c compounds were located and estimated by the l o c a l i n t e n s i t y of r a d i o a c t i v i t y .  The methods  c i t e d t o t h i s point have, i n general, estimated the amino-acids d i r e c t l y on the paper.  These methods have the disadvantage that  most u t i l i z e only one-directional separation of amino-acid which  16 i s often inadequate.  I f two-directional chromatography  i s used,  large numbers of chromatograms must be made i n order to obtain s a t i s f a c t o r y data.  This i s probably due to i n s u f f i c i e n t control  of the variable involved i n the chromatography.  Other methods  locate amino-acids on the paper, then remove the amino-acid from the paper and complete the quantitative estimation i n v i t r o . Martin and Mittelman (35) rejected both F o l i n ' s colour reaction and the ninhydrin r e a c t i o n f o r the quantitative determination of amino-acids a f t e r separation by paper p a r t i t i o n chromatography.  Micro-kjeldahl procedure and t i t r a t i o n o f the amino-  acids i n g l a c i a l a c e t i c a c i d were a l s o t r i e d and found wanting. They f i n a l l y dissolved the amino-acids from the paper, mixed them with insoluble copper phosphate and determined, polarographically, the copper which dissolved by forming a complex with the amino-acids. Woiwood (50) used t h i s same general procedure except that he measured the copper c o l o r i m e t r i c a l l y . In both these procedures, one-directional chromatograms only were used and the amino-acids must be located by simultaneous chromatography of known amino-acids. Woiwood(50) amplified h i s e a r l i e r work to give a more detailed procedure and has a l s o extended i t to include two-directional chromatography.  He used the copper phosphate procedure to test the n i n -  hydrin reaction, but came to some misleading  conclusions by the use  of too d i l u t e a s o l u t i o n of ninhydrin and by f a i l i n g to recognize that the reaction does not proceed once the paper i s dry. N a f t a l i n (39) and Awapora (4) located the amino-acids on  17  the paper by reaction with ninhydrin and then completed the colour development i n a teat tube. The method of Awapora (4) was modified by Landria and Axitapara (5).  This method consisted of the following.  Amino-acids were located on the paper with ninhydrin, the amino-acid spot was  cut out and the reaction was completed by heat-  ing i n a test tube with Moore and Stein's (48) ninhydrin s o l u t i o n which contained stannous chloride.  Although t h i s method gave high  colour production, i t has been u t i l i z e d only with u n i d i r e c t i o n a l chromatograms and gave e r r a t i c r e s u l t s , possibly because t h i s n i n hydrin s o l u t i o n reacted with ammonia.  Fowden and Penny (25) have  u t i l i z e d an analogous procedure except that the amino-acids were located by t h e i r flourescence i n the u l t r a v i o l e t l i g h t a f t e r heating the paper to 90 to 100° Centigrade.  They found that there were  losses of amino-acids unless the solvents were removed by washing with ether before treatment.  MATERIALS AND METHODS MATERIALS Field  Experiments The University 5-year blueberry planting consisted of 18  v a r i e t i e s with 5 plants of each v a r i e t y r e p l i c a t e d 9 times i n randomized blocks.  One t h i r d of the plants were mulched with a 2-inch  layer of peat, another t h i r d with a 2-inch layer of sawdust, and  IS  the remaining t h i r d was  clean cultivated. A l l p l o t s received  4 - 1 0 - 1 0 f e r t i l i z e r at the r a t e of 1000 pounds per acre. cluded i n the 18 v a r i e t i e s was  the v a r i e t y O o v i l l e .  tended to use t h i s v a r i e t y f o r l e a f and f r u i t Unfortunately  In-  I t was i n -  analyses.  a l l plants i n the f i e l d experiments were  dug up and removed by a University decree i n order to make 'Parking Lots'; thus depriving the w r i t e r of the use of these plants t o complete this- phase of the i n v e s t i g a t i o n . Greenhouse- Experiments One year old rooted cuttings of highbush blueberry  plants  var C o v i l l e - rooted i n peat - were taken d i r e c t l y from the propagating bed.  They were selected f o r uniformity of s i z e of both  top and root system. The tops consisted of 2 to 3 shoots of 20 to 25 cm. length.  in  The roots were washed c a r e f u l l y to remove as much of the  rooting medium as possible and the plants were set i n 10 inch clay pots containing quartz sand, one plant to each pot. was  The experiment  conducted i n one section of the University Greenhouses. On October 26, 1958,  one hundred and f i v e plants were set  up and arranged i n 3 blocks; each block consisted of 35 plants. Each block was  further divided into 7 plots (5 plants per p l o t ) ,  and l a b e l l e d A, B, C, 0, E, F, and  G.  A quantity of mixed sawdust ( f i r and hemlock) and a s i m i l a r amount o f peat was  used as a mulch and another p o r t i o n was  treated  19  at 22 p a i . f o r one hour.  Portions of the treated and untreated  peat and sawdust were placed i n different 10 g a l l o n earthenware crocks containing water and allowed to stand f o r s e v e r a l days; and these leachates were used f o r watering the p l a n t s . The experiment was set up i n the following manner: Plots  Treatments  A  Peat steam treated leachate.  B  Control - Khop*s s o l u t i o n .  C  Two inches o f sawdust were applied to the surface of the pots as a mulch,  D  Sawdust steam-treated leachate.  E  Peat untreated leachate.  F  Sawdust untreated leachate.  G  Two inches of peat were applied t o the surfaces of the pots as a mulch.  Weekly applications of 500 c.c. of Khop's nutrient s o l u t i o n were a p p l i e d to the surface o f each pot.  Besides the weekly  feedings of Khop's s o l u t i o n the mulched and control pots were kept moist with d i s t i l l e d water; t o the other pots; the various leachates were added r e p l a c i n g the d i s t i l l e d water.  In November, 1958, and  August, 1959, samples of percolates from each pot were taken and t h e i r pH values were determined with a Beckman pH meter. In August 1959, the accumulative height of each plant was again taken and recorded. In order to determine the effects of peat and sawdust  1  20  mulches on the dry weight, t o t a l mineral content (ash), t o t a l nitrogen and amino-acid content o f blueberry leaves, samples of the control s e r i e s (B), the sawdust mulched series (0) and the peat mulched series (G), were taken at random from the p l o t s i n each o f the three blocks.  Each sample was divided into two parts  and duplicate analyses were made on each p a r t . Moisture content was determined by weighing samples to a constant weight a f t e r drying i n an e l e c t r i c oven a t 70 degrees F. Ash determinations were made according to the standard method using a muffle furnace as set out i n the A.O.A.0. (3). T o t a l nitrogen was determined by the K j e l d a h l method ( 3 ) . A f t e r t r y i n g a number of the methods indicated i n the r e view o f l i t e r a t u r e , amino-acids were eventually determined by a onedimensional method o f paper chromatography as advocated by Dent, Stepe and Steward (29) with s l i g h t modifications.  Several other  workers (20, 34, 41, 38) recommend t h i s method i n preference t o other methods used i n s i m i l a r investigations.  Twenty-five grams of  the f r e s h l e a f material was placed In a porcelain mortar and ground with w e l l washed quartz sand to a homogeneous t h i n paste.  Ten c.c.  of 80 per cent e t h y l alcohol was added slowly to the sample during the grinding process.  This t h i n paste was then placed into c e n t r i -  fuge tubes and centrifuged f o r 10 minutes at the rate o f 500 revolut i o n s per minute.  The samples were removed from the centrifuge and  the supernatent l i q u i d was decanted l i q u i d was decanted  into clean test tubes.  into clean test tubes.  This  This l i q u i d was evaporated  21 to dryness, under reduced pressure, without the use of heat. To the residue was added 0.1 c.c. of d i s t i l l e d water. An 18  rt  x 22  M  sheet of Whatman no. 1 f i l t e r paper was  placed on a large sheet of brown paper on the top of the work bench, eare being taken to protect the f i l t e r paper from any contact with the hands or with the laboratory bench. A p e n c i l l i n e was drawn about 3 ^ d i c u l a r l i n e s at 2  B  n  from one end.  Along t h i s l i n e short perpen-  intervals were drawn. A t these i n t e r v a l s ,  using micro-pipettes, 10 m i c r o - l i t r e s of the solutions f o r analysis were applied, drop by drop, allowing each drop to dry before the successive one was added; so that a spot of about one centimetre i n diameter was obtained. As well as the solutions from the plants of the control, sawdust mulched and peat mulched p l o t s , 0.1 M solutions of alanine, asparagine, p r o l i n e , aspartic acid, glutamic acid and tryptophane were spotted. trials  These amino-acids were selected because preliminary  with several o f the methods outlined i n the review of  litera-  ture had indicated that they were present i n appreciable amounts. The spots were allowed t o dry and the sheets of f i l t e r paper were placed i n a chromatocab. A phenol-water^solvent consisting of 80 gm. phenol » 20 c.c.  water was applied to the trough and the chromatograms were  allowed to run f o r about 20 hours.  A f t e r t h i s time the chromato-  t  grams were removed from the chromatocab and allowed to dry by hanging  i n a fume hood and the Rf values were recorded.  22 The chromatograms were then sprayed with 0.555 ninhydrin solution (1, 2 , 3 -  triketohydrindene hydrate) i n isopropyl alcohol;  and incubated at 80° 0 to develop the c h a r a c t e r i s t i c amino-acid colour.  They were then photographed.  23 RESULTS OF EXPERIMENTS It was observed that the control plants at the end of the experiments were much smaller than those i n the p l o t s that were mulched or treated with the various leachates.  A l s o the  control plants showed a greater tendency to die back s t a r t i n g from the t i p s , than any of the treated plants.  These r e s u l t s agree  with the findings of Savage and Darrow (45). The l e a f surface area of control plants was much smaller than that o f the treated plants. Leaves of plants i n the control p l o t s were of a l i g h t green colour while the leaves o f the treated plants were a dark green colour. TABLE I The E f f e c t of Treatments on the pH of the percolates a f t e r passing through the growing medium. Blocks  Plots and Treatment  Nov. Aug. Nov. Aug. Nov. Aug. Nov. Aug. Nov. Aug. Nov. Aug. Nov. Aug. 1958 1959 1958 1959 1958 1959 1958 1959 1958 1959 1958 1959 1958 1959 5.8  5.8  5.8  5.8  5.8  5.8  5.9  5.8  5.8  5.8  5.7  5.8  5.9  5.8  5.8  5.9  5.8  5.9  6.0  5.9  5.8  5.9  5.8  5.7  5.8  5.8  5.7  5.7  5.9  5.8  5.9  5.8  5.8  5.7  5.9  5.7  5.8  5.7  5.8  5.9  5.8  5.7  Table I indicates that the plants throughout were feeding on a nutrient solution of approximately constant pH.  24 TABLE I I The e f f e c t o f peat and sawdust mulches, steam-treated and untreated leachates on the accumulative growth of blueberry plants (length of shoot measured i n centimetres). TREATMENTS  PEAT  SAWDUST  Mulch  Block 1 F i n a l Growth Original Growth Increase i n Growth  Leachate  CONTROL Leachate.  Mulch  G  Steam Treated A  Untreated E  781  516  52  i  C  Steam Treated D  Untreated F  B  356  680  430  394  345  49  74  58  54  58  55  729  467  282  632  376  336  290  867  537  418  605  552  552  334  85  61  65  66  57  69  67  782  476  353  539  495  483  267  802  534  479  748  462  351  474  65  58  62  78  60  34  60  737  476  417  670  402  287  414  Block I I F i n a l Growth Original Growth Increase i n Growth Block I I I F i n a l Growth Original Growth Increase i n Growth  25 TABLE I I I STATISTICAL ANALYSIS OF INCREASE IN SHOOT GROWTH ( i n Centimetres) Blocks  1 11 111 Treatment Totals  Plots and Treatments  Total Blocks  G  C  A  D  E  F  B  729 782 737  632 539 670  467 476 476  376 495 402  282 353 417  336 483 267  290 267 414  3112 3395 3383  2248  1841  1419  1273  1052  1086  971  9890  ANALYSIS OF VARIANCE Factor Total Treatments Blocks Error  S.S. 508877 444219 7318 57340  D.F. 20 6 2 12  I.S. 74036.5 3649.0 4778.3  E. 15.5 .79  0.05  Required F 0.01  3.00 3.89  4.88 6.93  In table I I I the calculated value of F f o r the treatments exceeds the required value of F.01 so that there are highly s i g n i f i c a n t differences among treatments. Table I I I also shows that the calculated value of F f o r the blocks i s lower than the required value of F.05; so that there are no s i g n i f i c a n t differences among blocks.  26 TABLE I I I A Treatments and accumulative mean shoot growth arranged i n Descending order  Or  c. A  D Untreated  Peat mulch Sawdust Mulch Steam treated peat leachate Steam treated sawdust leachate Sawdust leachate  749.3 603.6 473.0 426.3 362.0  Peat' leachate  350.7  Control  323.7  Centimetres n n if  »  •tf  Untreated  TS B  ISD./^  JSD. 01  =  2.179  /2 x 4778.5  =  2.179 x 56.421 = 122.9  =  3.055 x 56.421 = 172.3  Any treatment with an accumulative mean shoot growth greater than 446.6 centimetres i s s i g n i f i c a n t l y greater than the control (P  a  0.05).  The above data show that the treatments with peat mulch,  sawdust mulch, and steam-treated peat leachate s i g n i f i c a n t l y increased growth. Only peat and sawdust mulched plants showed a h i g h l y s i g n i f i cant increase i n growth over the control plants at the l e v e l of P.» 0.01. The peat mulch showed the greatest increase i n growth of a l l treatments.  27 TABLE 17  The e f f e c t o f sawdust and peat mulches on the average moisture, ash and t o t a l nitrogen content of the leaves o f blueberries, (figures represent averages of 12 determinations.)'  TREATMENT  MOISTURE  %  ASH  %  TOTAL NITROGEN  F.W.  %  F.W.  Control (Tjnmulched)  60.87  1.33  0.4005  Sawdust (Mulched)  60.43  1.51**  0.4320**  Peat (Mulched)  60.53  1.525**  0.4299**  ** - s i g n i f i c a n t l y greater than the control a t the l e v e l of P = 0.01. (see appendix)  Table 17 shows that the moisture affected by t h e treatments.  content of the leaves was not  Ash and t o t a l nitrogen were s i g n i f i c a n t l y  higher i n the leaves o f the plants from both the sawdust and peat mulched pots than i n the leaves of the plants from the unmulched pots.  There  was no s i g n i f i c a n t difference between the ash and t o t a l nitrogen content of the leaves from the sawdust mulched and peat mulched p l a n t s .  28 TABLE T  The Rf values and ninhydrin colour of amino-acids found i n the leaves of unmulched, sawdust mulched and peat mulched plants compared with standard values of c e r t a i n amino-acids.  Amino Acids  Colour  Aspartic acid  Blue  .05  —  —  —  Glutamic a c i d  Purple  .17  .20  .21  .20  Asparagine  Brown-purple  .33  .31  .32  .31  Proline  Yellow  .41  .40  .41  .40  Tyrosine  Blue  .48  .47  .46  .47  Alanine  Purple  #5^t  .52  .53  .52  Tryptophane  Brown-purple  .71  —  Phenylalanine  Blue  .83  .84  Standards  Rf Values Unmulched  Sawdust Mulched  -.83  Peat Mulched  —  .84  Table V shows that at the time of sampling the leaves of the unmulched, sawdust mulched and peat mulched plants had i n the free state amino-acids, alanine, glutamic a c i d , p r o l i n e , tyrosine, asparagine and phenylalanine • Although equal volumes o f the samples were spotted, the aminoacids present i n the unmulched plants covered a much larger area of the chromatogram than those present i n the sawdust or peat mulched plants. This indicates that the amino-acids i n the unpaulched plants were present in much greater amounts than i n the mulched p l a n t s .  29  Photograph, o f a t y p i c a l chromatogram comparing amino-acids i n samples from leaves of the unmulched and peat mulched plants i s shown i n F i g . I . FIGURE I  M —  Extracts from peat mulched p l a n t s .  U —  Extracts from unmulched p l a n t s .  u  Amino-Acids A - Glutamic Acid B - Asparagine C - Proline D - Tyrosine E - Alanine  30  TABLE 71 A r e l a t i v e evaluation of the nitrogen f r a c t i o n found i n the leaves of unmulched, peat mulched and sawdust mulched plants i n the form o f amino-acids  Amino-Acids  Unmulched  Mulched Sawdust Peat  Glutamic a c i d  Wt  W  W  Alanine  tW  w  ftt  Asparagine  W W  wt  W  Proline  Wtt  w  tt  Tyrosine  w  *•  t  Phenylalanine  W W  tt  tt  Average t o t a l  W+f  W  tt  Table 71 indicates t h a t the greatest accumulation of aminoacids was  found in the leaves of the unmulched plants, despite the  fact that the greatest t o t a l amount of nitrogen was leaves of the mulched plants (table I T " ) .  present i n the  31  D i s c i s s i o n of Results The b e n e f i c i a l e f f e c t s of the peat and sawdust mulches were pronounced* Under f i e l d conditions these benefits have been a t t r i b u t e d to f a c t o r s such as conservation of s o i l moisture, the maintenance of a more uniform s o i l temperature, and the provision of a more suitable pH of the growing medium.  However, under the uniform greenhouse con-  ditions where watering was adequately provided at a l l times, and the temperature remained f a i r l y constant, these factors were not l i m i t i n g * It would appear that in seme manner, the mulches i n question Increased the metabolic a c t i v i t i e s of the plants.  The increased a c t i v i -  ty was r e f l e c t e d by increased growth of the mulched plants, a higher ash content, and a higher t o t a l nitrogen content of the leaves. The pH values of the nutrient media ranged w i t h i n the l i m i t s of pH 5*7 - 6.0 f o r a l l treatments including the control*  The poor  growth of the control then, cannot be attributed to an unfavourable pH. Some workers (8, 19, 2 7 , 30) have suggested that a range o f pH 4.0  - 5.2  i s optimum f o r blueberries, and above or below t h i s pH range, blueberries may bot be expected to survive.  The present work does not support t h i s  contention, but rather, that blueberry plants may thrive equally w e l l at the higher pH ranges of pH 5 . 7 - 6.0 provided that an adequate nutrient supply i s maintained.  This i s i n agreement with B o i l e r (14)  and Cain ( 1 7 ) . The plants mulched with peat gave a better growth response than those mulched with sawdust.  It could be assumed that the benefic-  i a l effect of the peat was due to a lower C/N r a t i o i n the peat than i n the sawdust.  The sawdust would then tend to depress a v a i l a b l e nitrogen  32 in s o i l .  However, i n t h i s experiment  amounts i n a l l oases.  nitrogen was added i n adequate  The presence of growth hormone substances i n  the peat and not present, or present i n lesser amounts, i n the sawdust i s a p o s s i b i l i t y , but t h i s was  not investigated.  Plants mulched with sawdust showed a much greater growth than the control plants, or those to which leachates of peat or sawdust had been applied.  This b e n e f i c i a l effect of sawdust mulch on  blueberries agrees with the findings of Savage and Barrow  (45).  Plants to which steam.treated leachates were applied grew better than those aupplied ,with untreated leachates.  It i s possible  that the steam treatment softened the c e l l walls of the peat and sawdust and thus allowed f o r a more e f f i c i e n t e x t r a c t i o n of the water soluble n u t r i e n t s present i n the mulches, and so increased c e r t a i n nutrient concentrations a v a i l a b l e to the plant.  Further work on the  water soluble nutrients of peat and sawdust at various stages of decomposition, treated and untreated with steam, i s needed before a clear p i c t u r e of the factors contributing to the benefits of steam treatment can be formulated. The leaves of the mulched plants contained a higher percentage of t o t a l nitrogen but a lower concentration of amino-acids the leaves of the unmulched plants.  This may  than  indicate that the amino-  acids i n the mulched plants were combining more r e a d i l y than i n the unmulched plants t o form proteins. Six  amino-acids were i d e n t i f i e d .  Of these, two, v i z .  glutamic a c i d and alanine are known to play important roles i n p r o t e i n and carbohydrate metabolism. Glutamic a c i d i s probably synthesized i n plant cellB by a  33 reaction between ammonia, often o r i g i n a t i n g from the reduction of n i t r a t e s , and  eC-ketoglutarie a c i d , as follows:  COOH  COOH  (3=0  CHNHg  CHgWHj* DPN.Hg  *•  COOH  CH^H^OfDPN  COOH  <C- Ketoglutaric acid. Glutamic A c i d . This reaction i s catalyzed by the enzyme dehydrogenase, with diphosphopyredine mucleotide (DPN)  as the coenzyme.  i s widely and probably u n i v e r s a l l y prosent  i n plants.  This enzyme  Although de-  t a i l s of t h i s r e a c t i o n have been worked out only i n animal tissues (von E i l e r e t a l ) , there i s l i t t l e doubt that t h i s reaction a l s o occurs i n plant t i s s u e s .  This type of reaction i s known as reductive  amination. The other amino-acid of great s i g n i f i c a n c e found present i n the samples was alanine.  This a c i d may have been formed from  pyruvic a c i d by reactions analogous t o those stated above. A close i n t e r r e l a t i o n e x i s t s between the synthesis of c e r t a i n amino-acids, organic a c i d metabolism, and the process of aerobic r e s piration.  The r e v e r s i b l e reactions involving the conversion of  •C-ketoglutaric a c i d t o glutamic a c i d , alanine t o pyruvic a o i d , and other analogous reactions which may occur can be considered as side reactions of the t r i c a r b o x y l i c a c i d c y c l e . There i s considerable evidence that some of the amino-acids may  a r i s e as a r e s u l t of transmination reactions i n which amino groups  34  are transferred from one kind of a molecule t o another.  The follow-  ing i s an example o f such r e a c t i o n : COOH  COOH  CHNH  C aO transaminase  I  4-  CH3  Pyruvic a c i d  COOH  COOH  f  CBg  I  2  Alanine  COOH  COOH  rf-ketoglutaric acid  glutamic acid  Reactions of this kind are catalyzed by an enzyme of the type c a l l e d a transaminase.  Such enzymes are widely d i s t r i b u t e d in,  higher p l a n t s . Due  t o the above reactions glutamic acid and alanine may  be converted r e s p e c t i v e l y t o  eC-ketoglutaric acid and pyruvic a c i d  and here enter i n the t r i c a r b o x y l i c acid cycle i n carbohydrate metabolism. Thus i t may be assumed that these two amino-acids were r e acting more r a p i d l y i n the mulched plants than i n the unmulched plants; and thereby t h i s may account f a r t h e i r reduced amounts i n the mulched plants. This reaction, whereby these amino-acids enter the TCA cycle i n carbohydrate metabolizm, may also be correlated with the higher percentage In dry weight o f leaves i n plants from the mulched plots.  Assuming that the amino-acids, glutamic a c i d , and alanine  transaminate and deaminate to form  oC-ketoglutaric atfid and pyruvic  35  a c i d and thereby enter the t r i c a r b o x y l i c acid cycle, they would cause an increase i n the r e s p i r a t i o n rate and subsequently protein synthesis, and f i n a l l y increase the r a t e of growth. The better u t i l i z a t i o n of nitrogen i n the metabolic processes o f the blueberry as a r e s u l t of mulching i s of especial interest and warrants further investigation as to the mechanism involved.  36  CONCLUSIONS  From the r e s u l t s obtained, i t may be concluded that blueberry plants grow at a f a s t e r rate when mulched with sawdust or peat than when unmulched.  Free amino-acids are used up much f a s t e r  i n the mulched plants than i n the unmulched ones; r e s u l t i n g i n a higher p r o t e i n content i n the mulched plants than i n the unmulched plants. The application of mulches i n blueberry culture on mineral s o i l s can contribute t o increased y i e l d s .  Although only 6 amino-  acids were recognized, t h i s does not indicate that there are only 6 free ami no-acids present i n the blueberry plant. At the time of sampling translocation of amino-acids to various meristems may have occurred. The application of peat or sawdust mulches t o blueberry growing on mineral s o i l s would appear to be a sound c u l t u r a l p r a c t i c e .  37 SUMMARY  Experiments involving the use of C o v i l l e blueberry plants were conducted i n the f i e l d , greenhouses, and laboratories of the D i v i s i o n of Plant Science at the University of B r i t i s h Columbia to determine the e f f e c t s o f various mulches and t h e i r leachates on the growth and certain metabolic responsis of highbush blueberry plants. The r e s u l t s observed were as follows: (1)  Plants which had 2" of sawdust or peat applied as a mulch  showed a much greater increase In growth than the other treatments. (2)  The greatest Increase In growth was obtained from plants  which were mulched with peat. (3)  The least increase In growth was obtained from the control  plants, i . e . those which received water and nutrient s o l u t i o n . (4)  Steam treated sawdust leachate, untreated sawdust leachate,  and untreated peat leachate caused an increase i n growth but t h i s  was  not s i g n i f i c a n t . (5)  Plants to which the steam treated peat leachate was applied  showed a s i g n i f i c a n t increase i n growth over the control p l a n t s . (6)  There was l i t t l e v a r i a t i o n i n pH between various treatments  throughout the experiment. (7)  The pH ranged from 5.7  to 6.0.  Free amino-acids phenylalanine, proline, alanine, tyrosine,  glutamic acid and asparagine were found in both mulched and unmulched plants.  38 (8)  A much greater concentration of f r e e amino-acids  occurred i n the leaves of unmulched plants than i n those of the mulched plants. (9)  The percentage of oven d r i e d weight and ash content of  leaves from the mulched plants was higher than i n leaves from the unmulched plants. (10) The percentage of crude p r o t e i n i n leaves from the mulched plants was higher than i n leaves from the unmulched p l a n t s .  39 LITERATURE CITED  1.  Anderson, J.A. The influence of a v a i l a b l e nitrogen on the fermentation o f c e l l u l o s e i n the s o i l . S o i l S c i . 21: 115-126, 1926.  2.  A r i e s , R.S. The uses of l i g n i n as a s o i l builder. North eastern Wood U t i l i z a t i o n Council B u l l e t i n 19: 79-122. 1948.  3.  A s s o c i a t i o n o f O f f i c i a l A g r i c u l t u r a l Chemists. O f f i c i a l and tentative methods o f analyses. Washington.  4.  Awapora, J . Application of paper chromatography to the estimat i o n of some f r e e amino-acids i n tissue of the r a t . Jour. B i o l . Chem. 178: 113-116. 1949.  5.  1  and A.J. Landria. Use of modified ninhydrin reagent i n quantitative determination of amino-acids by paper chromatography. Sc. 109: 385, 1949.  6.  B a i l e y , J.S. and J.N. Everson. Further observations on chlorosis of the c u l t i v a t e d blueberry. Proc. Amer. Soc. Hort. S c i . 34: 495 - 9496. 1937.  7.  Beckwith, C.S. Blueberries i n the garden New Jersey Agr. Exp. Sta. C i r . 457. 1945.  8. 9.  , and C.A. Doehlert. F e r t i l i z e r and t i l l a g e f o r blueb e r r i e s , New Jersey Agr. Exp. Sta. B u i . 558. 1933. Berry, H.K. and I . Cain. Biochemical i n d i v i d u a l i t y . 17. A paper chromatographic technique f o r determining excretion of amino-acids in. the presence of i n t e r f e r i n g substances. Arch. Biochem. 34: 179-189. 1949.  10. Blasberg, C.H. Growing blueberries i n Oregon. Exp. Sta. Pamp. 19. 1948.  Oregon Agr.  11. Block, R.J. A comparative study on two samples of Neurokeratin Arch. Biochem.and Biophys. 31: 266-272. 1952. 12.  . Quantitative estimation of amino-acids on paper chromatograms. S c i . 109: 608-609. 1948.  13.  , B o i l i n g , D. and H.A. Sober. Quantitative separation and determination o f small amounts of h i s t i d i n e and tyrosine employing paper chromatography. Federal Proc. 8: 185. 1949.  40  14.  B o i l e r , C.A. Growing blueberries. Ore. Agr. Exp. S t a . B u l l . 499. 1951.  15.  B u l l , H.B., Hahn, J.W. and V.H. B a p t i s t . F i l t e r paper chromatography. Jour. Amer. Chem. Soc. 71: 550-553. 1949.  16.  Cain, J.C. Growth and development of blueberry f r u i t . Amer. Soc. Hort. S c i . 59: 167-173. 1952.  17.  Proc.  . Relationship between iron chlorosis of blueberry and the pH of l e a f t i s s u e . Abs. Amer. Soc. Hort. S c i . Sept. 1953.  18.  Chendler, F . B . E f f e c t of lime on low bush blueberry. Proc. Amer. Soc. Hort. S c i . 36: 477. 1939.  19.  C o l l i s o n , R.C. Making s o i l s a c i d f o r blueberries. New York Agr. Exp. Sta. Quart. B u i . 82: 8. 1942.  20.  Consden, R., Gordon, A.H., and A.J.P. Martin. Q u a l i t a t i v e analysis of proteins: A p a r t i t i o n chromatographic method using paper. Biochem. Jour. 38 : 224-232. 1944.  21.  Doehlert, G.A. F e r t i l i z i n g commercial blueberry f i e l d s i n New Jersey. New Jersey Agr. Exp. Sta. C i r , 483. 1944.  22.  , and J.W. Shive. N u t r i t i o n of blueberry i n sand cultures. S o i l S c i . 41: 341-350. 1936.  23.  Dunn, S. and J . S i e b e r l i c h . Uses of l i g n i n i n A g r i c u l t u r e . Mich. Eng. 67: 197-198. 1947.  24.  Fisher, R . B . , Parsons, D.S. and G.A. Morrison. Quantitative paper chromatography. Nature, 161: 765-765. 1948.  25.  Fowden, L. and J.R. Penny. Elimination of losses i n the quant i t a t i v e estimation of amino-acids by paper chromatography. Nature 165: 846-847. 1950.  26.  Griggs, B.H. and H.A. R o l l i n s . Effects of soil-management on y i e l d , growth, moisture, and ascorbic a c i d of blueberries. Proc. Amer. Soc. Hort. S c i . 51: 304-308. 1948.  27.  Harmer, P.M. The effects of varying the reaction; of organic s o i l on the growth and production of the domestic blueberry. Pro. S o i l . S c i . Soc. Amer. 9_: 133-141. 1944.  28.  Harris, G. Howell. Sawdust as a mulch f o r strawberries. S c i . A g r i . 31: 52-60. 1951  41  29.  Johnston, S. Influence of various s o i l s on growth and p r o d u c t i v i t y of highbush blueberry. Mich. Quart. Bui. 24: 307-310. 1942.  30.  Johnston, S. Behaviour of highbush and lowbush blueberry selections and t h e i r hybrids growing on various s o i l s located at different l e v e l s . Mich. Agr. Exp. Sta. Bui. 205. 1948.  31.  Johnston, W.A., and I.M. Ware. E f f e c t s of d i f f e r e n t mulches on s o i l a c i d i t y . Proc. Amer. Soc. Hort. S c i . 55: 285288. 1950.  32.  Keston, A.S., Udenfriend, S. and M. Levy. Paper chromatography applied to the i s o t o p i c derivative methods of analysis. Jour. Amer. Chem. Soc. 69: 31-51. 1947.  33.  Kramer, A. and A.L. Schrader. E f f e c t s of nutrient, media, and growth substances on the growth of Cabot variety of Vaceinium corymbosum L. Jour. Agr. Res. 65 : 313-328. 1942.  34.  Kramer, A. and A.I. Schrader. Significance o f pH o f blueberry leaves. Plant Phys. 20: 30-36. 1945.  35.  Martin, A.J.P. and R. Mittelman. Quantitative micro analysis of amino-acid mixtures on paper p a r t i t i o n chromatograms. Biochem. Jour. 43: 353-358. 1948.  36.  McHargue, J.S. The r o l e of manganese i n plants. Chem. Soc. 44: 1592-1598. 1922.  37.  Mikkelsen, D.S. and C.A. Dochlert. Magnesium deficiency i n blueberries. Proc. Amer. Soc. Hort. S c i . 55: 289-292. 1950.  38.  Moore, S. and W.H. S t e i n . Photometric ninhydrin method f o r use i n chromatography o f amino-acids. Jour. B i o l . Chem. 176: 367. 1949.  39.  N a f t a l i n , L. Quantitative chromatographic estimation of aminoacids. Nature. 161: 763. 1948.  40.  Perlmutter, F. and G.M. Darrow. E f f e c t of s o i l media, photoperiod and nitrogenous f e r t i l i z e r on the growth of blueberry seedlings. Proc. Amer. Soc. Hort. S c i . 40: 341346. 1942.  41.  Poison, A. Quantitative p a r t i t i o n chromatography and the composition of E. c o l i . Biochem. et Biophys. Acta. 2: 575-581. 1948.  Jour. Amer.  42 42.  , Moselay, V.M. and R.W.C. Wyckoff. chromatography o f s i l k hydrolysate. 604. 1947.  The quantitative Science 105: 603-  43.  Pope, C.C. and M.F. Stevens. Microestimation o f aminonitrogen and i t s application of paper p a r t i t i o n chromatography. Biochem. Jour. 33: 1070. 1939.  44.  Rockland, L.B. and M*S. Dunn. Quantitative determination of amino-acids on f i l t e r paper chromatograms by d i r e c t photometry. Jour. Amer. Chem. Sec. 71: 4121-412&. 1949.  45.  Savage, E.E. and G-.M. Darrow. Responses o f blueberries under clean c u l t i v a t i o n and various kinds of mulch materials. Proc. Amer. Soc. Hort. S c i . 40: 338-340. 1942.  46.  Shutak, V.G-. and E.P. Christopher. Effects of various c u l tured practices on the growth and y i e l d o f blueberries, Proc. Amer. Soc. Hort. S c i . 57: 64. 1951.  47.  Turk, L.M. The effect of sawdust on plant growth. Expt. Sta. Quar. Bui. 26: 10-22. 1948.  48. 49.  Mich. Agr.  , and N.L. Partridge. E f f e c t s of various mulching materials on orchard s o i l s . S o i l S c i . 64: 111-125. Woiwood, A.J. Micro-estimation of amino-nitrogen and i t s application to paper p a r t i t i o n chromatography, Nature 161: 169. 1948.  43 APPENDIX I Analysis o f the effect o f sawdust and peat mulches on the moisture content of the leaves of blueberry plants (each figure for the samples represents the average of 2 determinations).  B = Control, C • Sawdust mulched plants, G r Peat mulched plants.  SAMPLES  B  TREATMENTS C  G  TOTALS  1  60.82  60.81  60.00  181.63  2  60.85  60.84  60.00  181.69  3  60.85  60.83  60.03  181.71  4  60.91  60.90  60.11  181.92  5  60.92  60.94  60.05  181.91  60.92  60.91  60.14  181.97  365.27  365.23  360.33  6 TREATMENT TOTALS  1090.83  T o t a l S.S. = 60.82 f 60.85 4- ... 60.05 f 60.14 s  2  2  2  - 1090.83 = 3.29 2  Between Treatments S.S. r 565.27 * 565.23 f 560.33 - 1090.85 2 . 7 1 6 18 2  s  8  s  a  Error S.S. = 3.29-2.71  s  .58  Analysis of Variance Factor  S.S..  D.F.  M.S.  F.  Required f 0.05 0.01  3.57  3.68  Total  3.29  17  Between Treatments  2.71  2  1.355  .58  15  .38  Error  ^.05  2.131  .38 x 6 x 2  =  4.62  3.36  44 APPENDIX I I  A n a l y s i s of the effect o f sawdust and peat mulches on the t o t a l mineral (ash) content of the leaves of blueberry plants.  (Each figure f o r the samples represents the average o f  2 determinations). B » Control,  C = Sawdust mulched plants,  B  TREATMENTS C  G  TOTALS  1.29 1.28 1.28 1.37 1.36 1.38  1.53 1.51 1.54 1.50 1.55 1.50  1.52 1.52 1.52 1.53 1.53 1.54  4.34 4.31 4.34 4.40 4.44 4.42  7.96  9.13  9.16  26.25  SAMPLES 1 2 5 4 5 6 TREATMENT TOTALS  Total S.S. = 1.29  3  + 1.28  2  + 1.28 —1.53  Between treatments S.S. = 7.96  Error S.S.  G =• Peat mulched plants  =  s  s  4- 9.15 6  s  + 1.53  s  f 9.16  8  * 1.54  s  - 26.25 18  L S D . =  2*131  - 26.25 = .17 .16  s s  .17 - .16 = .01  Analysis of Variance Factor Total Between Treatments Error  s  S.S. .17 .16 .01  .0007x6x2  D.F. 17 2 15  .  M.S. .08 .0007  .192  F 114  Required f 0.01 a.©5 3.68  6.36  45 APPENDIX I I I A n a l y s i s of the e f f e c t of sawdust and peat mulches on the t o t a l nitrogen content of the leaves o f blueberry plants. figure f o r the samples represents the average of 2  (Each  determinations).  B = Control, C = Sawdust mulched plants, G = Peat mulched p l a n t s .  SAMPLES  TREATMENTS C  B .4005 .4005 .4006 .4006 .4007 .4007  .4389 .4382 .4390 .4212 .4211 .4210  .4340 .4340 .4338 .4300 .4301 .4302  1.2734 1.2727 1.2734 1.2518 1.2519 1.2516  2.4033  2.5794  2.5921  7.5748  1 2 3 4 5 6 TREATMENT TOTALS  TOTALS  G  Total S.S. - .4005 4- .4005 f ••••4301 f .4302 - 7.5748 18 2  2  2  Between Treatments S.S. = 2.4033 f 2.5794 6 2  Error S.S.  a  2  2  f 2.5921  Total Between treatments Error LSD.  05  =  2.131  S.S.  D.F.  M.S.  .0041 .0037 .0004  17 2 15  .00185 .00003  .0003x6x2  = .0041  - 7.5748 18  2  r .0037  .0041 - .0037 Z .0004  Analysis of Variance Factor  2  2  »  .12786  F 61.67  Required t .01 .05 3.68  6.36  

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