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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 (Vaccinium corymbosum L. var 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 accept t h i s t h e s i s as conforming to the standard requirement from candidates f o r the Degree of Master of Science i n Ag r i c u l t u r e • Members of the D i v i s i o n of Plant Science THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1 9 6 1 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Br i t i s h Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that per-mission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his represen-tatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Horticulture The University of British Columbia, Vancouver 8 , Canada. Date A p r i l , 1961. ABSTRACT Greenhouse, f i e l d and laboratory experiments were conducted to ascertain some growth and metabolic responses of the Coville blueberry variety, to sawdust and peat mulches, and the leachates of these mulches. From the greenhouse experiments i t would appear that in some manner the mulches i n question increased the metabolic ac t i v i t i e s of the plants. The increased ac t i v i t y was reflected by increased growth of the mulched plants, a higher ash content and a higher total nitrogen content of the leaves. A greater concentration of free amino acids ocourred in the unmulched plants than i n the mulched ones; This indicates that the nitrogen metabolic a c t i v i t i e s were hastened by the mulching. AOKMOWLEDGrEMEKTB The writer wishes to express his thanks to Dr. G, H. Harris, Professor of Horticulture, whose technical advice, time spent, and information offered was of inestimable value in the preparation of this thesis. TABLE OF CONTENTS Introduction 1 Review of Literature 3 Mater ialB and Methods 17 Fi e l d Experiments 17 Greenhouse Experiments 13 Results of Experiments 23 Table I . . , The Effect of Treatments on the pH of the percolates after passing through the growing medium 23 Table II The effect of peat and sawdust mulches, steam-treated and untreated leachates on the accumulative growth of blueberry plants (length of shoot measured i n centi-metres) 24 Table III Sta t i s t i c a l Analysis of Increase in Shoot Growth (in Centimetres) 25 Table III A Treatments and accumulative mean shoot growth arranged in Descending order 26 Table 17 The effect of sawdust and peat mulches on the average moisture, ash and total nitrogen content of the leaves of blueberries, (figures represent averages of 12 determinations.) 27 Table Y 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 certain ami no-a c ids 28 Figure I Photograph of a typical chromatogram comparing amino-aclds in samples from leaves of the unmulched and peat mulched plants 29 Table VI A relative evaluation of the nitrogen fraction found in the leaves of unmulched, peat mulched and sawdust mulched plants in the form of amino-acids 30 Page Discussion of Results 31 Conclusions 3 6 Sunmjaryu. 37 Literature cited ..; 39 Appendix % 43 Appendix II 44 /•.rsAppendix III 45 1 IMTRODTJCTION Experiments i n blueberry culture were initiated by F.V. CoviUe i n 1906. He selected and crossed wild species of high and low bush form and from 1921 to 1939 produced 18 named varieties. In 1949 these 18 varieties constituted the entire commercial acreage of highbush blueberries in America with the exception of Rubel, a variety selected from the wild by Miss E.C. White of Whitesborg, New Jersey. At his death in 1937 Dr» Ooville l e f t many thousands of seedlings which have given rise to further superior varieties. Today there i s a total of about 4,000 acres of the cultivated highbush blueberry i n the United States. In Canada the industry is small but is rapidly expanding i n the Maritimes, Ontario, and British 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 of the ava i l a b i l i t y of large acreages i n Bri t i s h Columbia potentially suitable for blueberry production, together with a brisk and increasing demand for the f r u i t , i t was f e l t that a contribution in the f i e l d of blueberry nutrition would be of value in fostering this neophyte B.C. Industry. In certain blueberry growing areas the cultivated blue-berry appears to suffer from various nutritional disorders. Poor growth has been attributed to such factors as magnesium, iron and 2 manganese deficiencies, unfavourable pH and nitrogen relation-ships. A number of remedies have been tried with varying degrees of success. On mineral soils the use of mulching materials has in certain cases, stimulated growth and improved the health and vigour of the plants. It was decided to investigate the effect of peat and sawdust mulches on the growth and vigour of the blueberry. Further-more, as an indication of the nutritional status of the plants i t was considered adviseable to ascertain the effect of the mulches on the moisture content, total mineral content (ash) and to t a l nitrogen content of the leaves. It was also f e l t that a study of the free amino-acids in the leaves would be helpful i n understand-ing the metabolic activity of the plant. 3 REVIEW OF LITERATURE Some Effects of Mulches on Blueberries, A comparison of the effect of sawdust and straw mulches with a buckwheat cover crop and clean cultivation was made by Shutak and Christopher (46) i n Hew Jersey using the Pioneer variety of blueberry. They found that consistently higher yields were produced by bushes mulched with sawdust as contrasted to clean cultivated plots which produced the lowest yields. 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 temper-atures were lower i n spring and summer and higher i n the f a l l under the mulch. A larger and mare fibrous root system developed under the sawdust mulch than with the other treatments. These workers also reported that weed control was greatly simplified by the use of the sawdust mulches. Chandler (18) reported that under conditions in Maine, the s o i l mulched with sawdust under blueberry plantings, retained more moisture and maintained lover temperatures than in unmulched plantings. The mulch increased growth of plants in clay and loam s o i l s , but on the other hand reduced growth of 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 cultiva-tion, but not as good as sawdust. Kramer (33) i n Maryland reported increased yields of both high and dryland blueberries were obtained with various mulches 4 although mulching decreased survival of the dryland blueberries. Mulching with sawdust in Connecticut gave greater growth and better yields than either clean cultivation or hay mulch accord-ing to Griggs and Rollins (26). The different types of s o i l manage-ment however did not affect the moisture content or the ascorbic acid content of the berries* Several workers (2, 23, 47, 48) have reported that fresh sawdust depressed available s o i l nitrogen, but that rotted sawdust was an excellent s o i l addition. Johnston (29) found that sawdust alone used as a surface mulch depressed nitrates i n the s o i l slightly for 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 for 18 months after which time they accumulated. According to Turk (47), i n Michigan an average sawdust contains 4 lbs. of nitrogen, 2 lbs. phosphorous pentoxide and 4 lbs. of potash per ton of dry material. This is about one-third the amount of nutrients i n the same weight of wheat straw. Comparing the effects of clean cultivation, sawdust mulch, oak leaf mulch, and rye straw mulch on blueberry plants, Savage and Darrow (45) found the plants from mulched plots were superior to those from clean cultivated plots and that sawdust was superior to either oak leaves or rye straw. On the basis of plants surviving at the end of the th i r d year the sawdust mulch was superior to rye straw 5 and oak leaf mulch. The plants In the clean cultivated plots were so small that, ccmnercially their productive capacity was regarded as valueless* Anderson (1) reported that the addition of straw and cellulose depressed available? nitrogen in 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 to leaching losses i n the un-mulched plots, the total nitrate accumulation was the same in a l l plots at the end of his experiment. Shutak (46) found that sawdust mulches increased the water holding capacity of the s o i l i n blueberry plots but that the increase was not as great as when straw mulch was used. Turk (47) reported that the effect of sawdust i s largely physical, resulting in a better water holding capacity, decreased s o i l losses due to erosion and the prevention of a hard crust on the s o i l surface limiting the capillary 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 effects of the increased mois-ture holding capacity of the s o i l due to the mulch Harris (28) pro-vided uniform moisture i n a l l plots with irrigation and found that a sawdust mulch markedly increased the yields of strawberries in a sandy s o i l compared to that of unmulched plants* 6 Blueberry Nutrition, Doehlert and Shive (22) working with both sand cultures and f i e l d plot experiments to determine the nutritional needs of the cultivated blueberry, (Vaccinium corymbosum), reported that the best nutrient solutions for the blueberry were low in phosphorus and high in nitrogen. Nitrate nitrogen appeared to be of greater value than ammonium nitrogen for this particular species. Good agreement was obtained between the results found in the sand cultures and those found in the f i e l d plots. The blueberry was shown to be sensitive to deficiency of boron and manganese. Cain (16) reported that aamonium nitrogen was superior to nitrate nitrogen for blueberries i n contrast to the results found by Doehlert and Shive (22). Plants which received nitrate nitrogen showed iron deficiency symptoms more readily than those which received ammonium nitrate although the iron content of the leaves in both cases was similar. He concluded that ammonium nitrogen was i n some way associated with iron nutrition. Perlmutter and Darrow (40) under the conditions of their 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 also concluded that s o i l pH may not necessarily be a direct controlling factor in iron absorption because plants growing on soi l s of relatively high pH often contained as much iron i n their foliage as those growing at a lower pH. In a study of a chlorotic condition of blueberry leaves, Mikkelsen and Loth (37) found that green leaves contained 350 ppm. 7 of soluble magnesium as compared with 30 ppm in the chlbrotic leaves. McHargue (36) reported that manganese is concerned i n nitrogen assimilation and i n the synthesis of proteins and that i t functions as a catalyst in plant metabolism and also functions with iron in 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 acidity for blueberry growth, suggesting a range of pH of 4.0 to 5.2 above or below which blueberries may not be expected to survive. Boiler (13), however claimed that s o i l structure, aeration, organic matter and water are the most important factors i n blueberry culture and that i f these conditions are sa t i s -factory, plantings could be maintained over a wide pH range. Cain (17) concurred with Boiler and reported successful plantings on soils above pH 6.0 i n the f i e l d and at pH 6.5 in nutrient solutions when an adequate nutrient supply was maintained. He con-sidered that s o i l pH was not necessarily a direct controlling factor in iron absorption because plants growing on s o i l s of relatively high pH often contained as much iron in their foliage as those growing at a lower one. Spraying the foliage with soluble iron salts was recommend-ed by Blasberg (10) to correct chlorosis associated with high pH s o i l s , but only as a temporary corrective measure because the chloro-sis usually reappeared i n new growth made after the spray application. 8 Bailey and Everson (6) corrected chlorosis with s o i l applica-tions of ammonium sulphate or aluminum sulphate. T hey found soluble iron available in the s o i l as a result of their treat-ments. Beckwith (7) suggested that ammonium sulphate gave better growth of blueberry plants i n soi l s above pH 5.5 and Doehlert (21) recommended its use i n connection with lime on commercial blueberry plantings. Johnston and Ware (31) performed experiments using saw-dust 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 in Blueberry Nutrition. McHargue (36) reported that manganese i s concerned in nitrogen assimilation and i n the synthesis of proteins; and that i t functions as a catalyst i n plant metabolism and also functions with iron i n the synthesis of chlorophyll. He considered also thaft-. since low manganese affects the production of dry matter, the i n -dications are that i t has some effect upon carbon assimilation. Typical symptoms of manganese deficiency in plants are:-the terminal bud remains alive and leaves do not wilt; chlorosis i s present with spots of necrotic tissue scattered over the leaf; the small veins tend to remain green giving the leaves a checkered or reticulated effect (36). On the other hand manganese excess i s typified i n i t i a l l y by a yellowing of the leaves accompanied by slight upward curling of the upper leaves. The chlorosis continues u n t i l the newer 9 leaves are almost white. The leaves then curl down and the mid-ribs often darken and break down. Large necrotic areas f i n a l l y develop in the chlorotic leaves and this i s accompanied by the breakdown and death of the stem (36). Magnesium i n Blueberry Nutrition. Mikkelsen and Loth (37) established the fact that the soluble magnesium contents of green and chlorotic leaves were different. Normal green leaves contained 350 ppm of magnesium as compared with 30 ppm. in the chlorotic leaves. S o i l samples co l l e c t -ed near affeoted plants contained only a trace of available magnesium. Kramer and Schrader (33) studied the effects of mineral deficiencies on the growth of the Cabot blueberry in sand cultures. They described magnesium deficiency as a marginal leaf chlorosis of the basal leaves extending intra-veinally with green midrib. The late stages of this deficiency are 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 varieties in New Jersey, except that necrosis has been rare. Very few data are available on the rates of s o i l applica-tion of magnesic f e r t i l i z e r s necessary to correct magnesium de-ficiency. Mikkelsen and Doehlert (37) reported that magnesium deficiency in 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 salts and hydrated dolomitic lime, respectively, 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 milliequivalent cation composition of them, while phosphorus, iron and manganese composition was relatively unaffected. The foregoing indicates that there i s disagreement i n the value of mulches used in blueberry culture and that the evid-ence conflicts as to the various causes of poor growth. The following i s a review of chromatography methods used for the detection of free amino-acids. Qualitative Paper Partition 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 allow-ing a suitable solvent, which has previously been saturated with water to flow over the paper in a closed container, the air 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 con-ditions, and separation depends upon the differences i n partition co-efficient of the amino-acids between the stationary water phase and the moving solvent. It has further been shown that for any individ-ual amino-acid the value: Rf = distance moved by the amino-acid distance moved by the advancing front of liquid i s directly related to the partition coefficient, true absorption by the cellulose playing l i t t l e part. Qualitative Analysis of Amino-acids. In an experiment on the qualitative micro-analytical tech-nique for amino-acids, Consden, Gordon and Martin (20) reported on: 1. Rate of diffusion of Amino-acids i n various solvents. 11 A number of solvents have been tried and abandoned. Of these ethyl acetate, methylethyl ketone, aniline, cyclohexanol, cyclohexane, quinoline and a light aniline fraction b.p. 174-180 were unsatisfactory as the amino-acids ran too slowly, whereas with methyl acetate, acetone, sec-butanol, pyridine, the picolines and the lutidines, the bands of amino-acids either moved too fast or were unduly broadened. Hydroxy-amino-acids move more slowly than the corresponding amino-acids in phenol, but i n collidine the rates are similar. Ammonia selectively slows the movement of aspartic and glutamic acids and hastens that of basic amino-acids. Acid has the reverse effect. 2. Additions of Acids Bases and Salt Hydrolysates, or mixtures in which the ratio of soluble inorganic salts of amino-acid were high, gave unsatisfactory chromatograms. The salt absorbed water from the atmosphere and the solvent and caused local water-logging of the paper. The amino-acids were not readily washed from these regions and the resulting bands were grossly distorted. This salt effect was eliminated by impregnating the paper with salt and using the solvent and atmosphere equilibrated with saturated salt solu-tion instead of with water. Micro Estimation of Amino-nitrogen. Pope and Stevens (43) described a method for determining amino and peptide nitrogen. Woiwood (50) successfully adapted this method to the determination of micro amounts ( 1 - 2 5 ugm) of eC -amino-nitrogem. The essential points of the method were that after 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 characteristic golden colour was extracted in amyl alcohol and the amount of copper determined absorptiometrically. The theoretical value of 0.44 for the ratio of fi- 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 for each individual amino-acid. Such curves were reproducible, and over their linear por-tion (10 - 25 ugm, «C- amino-nitrogen) the amino-acids methionine, hydroxyproline, aspartic acid, leucine, isoleucine, histidine, phenylalanine, valine, glutamic acid, tryptophane and tyrosine gave ratios lying between 0.50 and 0.55; alanine, glycine, threonine, serine, cystine and cysteine ratios between 0.55 and 0.80; and lysine, arginine and glucosamine ratios between 12.5 and 1.6. Quantitative Paper Chromatography. Quantitative Application of Paper Chromatography to Amino -Acids. One of the two major problems i n developing a method for quantitative determination of amino-acids by paper partition chroma-tography is the measurement of the amino-acids after they have been chromatographed. A number of procedures have been proposed which include colorimetric and polarographic methods as well 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 for determining amino-acids after separa-tion by paper partition 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 fact that the acetone is a poor solvent for the coloured product, these authors apparent-l y found other disadvantages to this procedure because i t was ab-andoned for one in 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 size of coloured areas. Fisher et a l (24) measured the area of the spot formed by the ninhydrin reaction, and found that the size was proportional to the logarithm of the con-centration of the amino-acid. The concentration of an amino-acid in a sample can then be determined by comparison with standards run simultaneously. One technique they used was a photographic method for determining a spot size. Bull et a l (15) determined the light transmission of the coloured compound on the paper at different points along the spot 14 and i n the direction of phenol movement. By integrating the area under the curve relating colour density and distance, an estimate of quantity was made. Block (12) used a similar method except that the amino-acids were f i r s t separated into acidic, basic, and neutral amino-acids on ion exchange resins before chromatography. 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 this method further in order to obtain the percentage composition of an amino-acid mixture. The method is more adapted to relative rather than absolute values. The maximum intensity 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 for each amino-acid was determined at the same molar concentration. The ratio of the colour density for a particular amino-acid to the sum of the colour densities of a l l the amino-acids con-stitute a "standard colour ratio for the amino-acid in question". An "experimental colour ratio?'for each amino-acid is determined in 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 ratio" gives the molar ratio of each amino-acid in the mix-ture. Analysis of casein by the empirical method agreed closely with published values although a large number of chromatograms (11) 15 were necessary to achieve this, Rockland and Dunn (44) used a densitometrie method for 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 difference. In the analysis of amino-acids i n urine by chromatography, Berry and Cain (9) u t i l i z e d visual comparisons of spots. Owing, however, to the effect op. the chromatography of 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 of their methods, the recovery of added acid varied over as wide a range as 63 to 160 percent, A novel method somewhat analogous to that of Bull 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 resulting compounds were chromatographed i n n-pentanol saturated with 2N ammonium hydroxide. The radioactivity was measured i n the direction of solvent movement, and specific compounds were located and estimated by the local intensity of radioactivity. The methods cited to this point have, in general, estimated the amino-acids directly 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 is often inadequate. I f two-directional chromatography is used, large numbers of chromatograms must be made in order to obtain satisfactory data. This is probably due to insufficient control of the variable involved in 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 Folin's colour reaction and the ninhydrin reaction for the quantitative determin-ation of amino-acids after separation by paper partition chroma-tography. Micro-kjeldahl procedure and t i t r a t i o n of the amino-acids i n gla c i a l acetic acid were also tried 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 this same general procedure except that he measured the copper colorimetrically. 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 his earlier work to give a more detailed pro-cedure and has also extended i t to include two-directional chroma-tography. He used the copper phosphate procedure to test the nin-hydrin reaction, but came to some misleading conclusions by the use of too dilute a solution of ninhydrin and by f a i l i n g to recognize that the reaction does not proceed once the paper is dry. Naftalin (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 solution which contained stannous chloride. Although this method gave high colour production, i t has been u t i l i z e d only with unidirectional chromatograms and gave erratic results, possibly because this nin-hydrin solution reacted with ammonia. Fowden and Penny (25) have util i z e d an analogous procedure except that the amino-acids were located by their flourescence in the ultraviolet light after heat-ing 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 Fie l d Experiments The University 5-year blueberry planting consisted of 18 varieties with 5 plants of each variety replicated 9 times i n ran-domized blocks. One third of the plants were mulched with a 2-inch layer of peat, another third with a 2-inch layer of sawdust, and IS the remaining third was clean cultivated. A l l plots received 4 - 1 0 - 1 0 f e r t i l i z e r at the rate of 1000 pounds per acre. In-cluded in the 18 varieties was the variety Ooville. It was i n -tended to use this variety for leaf and f r u i t analyses. Unfortunately a l l plants in the f i e l d experiments were dug up and removed by a University decree in order to make 'Park-ing Lots'; thus depriving the writer of the use of these plants to complete this- phase of the investigation. Greenhouse- Experiments One year old rooted cuttings of highbush blueberry plants var Coville - rooted i n peat - were taken directly from the pro-pagating bed. They were selected for uniformity of size of both top and root system. The tops consisted of 2 to 3 shoots of 20 to 25 cm. i n length. The roots were washed carefully 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. The experiment was conducted in one section of the University Greenhouses. On October 26, 1958, one hundred and five 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 plot), and labelled A, B, C, 0, E, F, and G. A quantity of mixed sawdust ( f i r and hemlock) and a similar amount of peat was used as a mulch and another portion was treated 19 at 22 pai. for one hour. Portions of the treated and untreated peat and sawdust were placed i n different 10 gallon earthenware crocks containing water and allowed to stand for several days; and these leachates were used for watering the plants. The experiment was set up i n the following manner: Plots Treatments A Peat steam treated leachate. B Control - Khop*s solution. C Two inches of 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 to the surfaces of the pots as a mulch. Weekly applications of 500 c.c. of Khop's nutrient solu-tion were applied to the surface of each pot. Besides the weekly feedings of Khop's solution the mulched and control pots were kept moist with d i s t i l l e d water; to the other pots; the various leachates were added replacing 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 their 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), total nitrogen and amino-acid content of blueberry leaves, samples of the control series (B), the sawdust mulched series (0) and the peat mulched series (G), were taken at random from the plots i n each of the three blocks. Each sample was divided into two parts and duplicate analyses were made on each part. Moisture content was determined by weighing samples to a constant weight after drying in an electric oven at 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). Total nitrogen was determined by the Kjeldahl method (3). After trying a number of the methods indicated in the re-view of literature, amino-acids were eventually determined by a one-dimensional method of paper chromatography as advocated by Dent, Stepe and Steward (29) with slight modifications. Several other workers (20, 34, 41, 38) recommend this method i n preference to other methods used in similar investigations. Twenty-five grams of the fresh leaf material was placed In a porcelain mortar and ground with well washed quartz sand to a homogeneous thin paste. Ten c.c. of 80 per cent ethyl alcohol was added slowly to the sample during the grinding process. This thin paste was then placed into centri-fuge tubes and centrifuged for 10 minutes at the rate of 500 revolu-tions per minute. The samples were removed from the centrifuge and the supernatent l i q u i d was decanted into clean test tubes. This liq u i d was decanted into clean test tubes. This liquid 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 18rt 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 con-tact with the hands or with the laboratory bench. A pencil li n e was drawn about 3 ^ n from one end. Along this line short perpen-dicular lines at 2 B intervals were drawn. At these intervals, using micro-pipettes, 10 micro-litres of the solutions for 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 in diameter was obtained. As well as the solutions from the plants of the control, sawdust mulched and peat mulched plots, 0.1 M solutions of alanine, asparagine, proline, aspartic acid, glutamic acid and tryptophane were spotted. These amino-acids were selected because preliminary t r i a l s with several of the methods outlined in the review of l i t e r a -ture had indicated that they were present i n appreciable amounts. The spots were allowed to dry and the sheets of f i l t e r paper were placed in 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 for about 20 hours. After this time the chromato-t grams were removed from the chromatocab and allowed to dry by hang-ing 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 characteristic 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 in the plots that were mulched or treated with the various leachates. Also the control plants showed a greater tendency to die back starting from the t i p s , than any of the treated plants. These results agree with the findings of Savage and Darrow (45). The leaf surface area of control plants was much smaller than that of the treated plants. Leaves of plants i n the control plots were of a light green colour while the leaves of the treated plants were a dark green colour. TABLE I The Effect of Treatments on the pH of the percolates  after 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 II The effect of 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 CONTROL Mulch Leachate Mulch Leachate. i G Steam Treated A Untreated E C Steam Treated D Untreated F B Block 1 Final Growth 781 516 356 680 430 394 345 Original Growth 52 49 74 58 54 58 55 Increase i n Growth 729 467 282 632 376 336 290 Block II Final Growth 867 537 418 605 552 552 334 Original Growth 85 61 65 66 57 69 67 Increase i n Growth 782 476 353 539 495 483 267 Block III Final Growth 802 534 479 748 462 351 474 Original Growth 65 58 62 78 60 34 60 Increase i n Growth 737 476 417 670 402 287 414 25 TABLE III STATISTICAL ANALYSIS OF INCREASE IN SHOOT GROWTH (in Centimetres) Blocks Plots and Treatments Total Blocks G C A D E F B 1 729 632 467 376 282 336 290 3112 11 782 539 476 495 353 483 267 3395 111 737 670 476 402 417 267 414 3383 Treatment 2248 1841 1419 1273 1052 1086 971 9890 Totals ANALYSIS OF VARIANCE Factor S.S. D.F. Total 508877 20 Treatments 444219 6 Blocks 7318 2 Error 57340 12 I.S. 74036.5 3649.0 4778.3 E. 0.05 15.5 3.00 .79 3.89 Required F 0.01 4.88 6.93 In table III the calculated value of F for the treatments exceeds the required value of F.01 so that there are highly significant differences among treatments. Table III also shows that the calculated value of F for the blocks i s lower than the required value of F.05; so that there are no significant differences among blocks. 26 TABLE III A Treatments and accumulative mean shoot growth arranged in Descending order Or Peat mulch 749.3 Centimetres c . Sawdust Mulch 603.6 n A Steam treated peat leachate 473.0 n D Steam treated sawdust leachate 426.3 if Untreated •tf Sawdust leachate 362.0 » Untreated TS Peat' leachate 350.7 B Control 323.7 ISD./^ = 2.179 /2 x 4778.5 = 2.179 x 56.421 = 122.9 JSD. = 3.055 x 56.421 = 172.3 01 Any treatment with an accumulative mean shoot growth greater than 446.6 centimetres i s significantly 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 significantly increased growth. Only peat and sawdust mulched plants showed a highly s i g n i f i -cant increase in growth over the control plants at the level of P.» 0.01. The peat mulch showed the greatest increase in growth of a l l treatments. 27 TABLE 17 The effect of sawdust and peat mulches on the average moisture, ash and total nitrogen content of the leaves of 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** ** - significantly greater than the control at the level of P = 0.01. (see appendix) Table 17 shows that the moisture content of the leaves was not affected by the treatments. Ash and total nitrogen were significantly higher i n the leaves of the plants from both the sawdust and peat mulched pots than in the leaves of the plants from the unmulched pots. There was no significant difference between the ash and total nitrogen content of the leaves from the sawdust mulched and peat mulched plants. 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 com-pared with standard values of certain amino-acids. Amino Colour Standards Rf Values Sawdust Peat Acids Unmulched Mulched Mulched Aspartic acid Blue .05 — — — Glutamic acid 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 .83 .84 Table V shows that at the time of sampling the leaves of the unmulched, sawdust mulched and peat mulched plants had in the free state amino-acids, alanine, glutamic acid, proline, tyrosine, asparagine and phenylalanine • Although equal volumes of the samples were spotted, the amino-acids present in the unmulched plants covered a much larger area of the chromatogram than those present in the sawdust or peat mulched plants. This indicates that the amino-acids i n the unpaulched plants were present in much greater amounts than in the mulched plants. 29 Photograph, of a typical chromatogram comparing amino-acids in samples from leaves of the unmulched and peat mulched plants i s shown in Fig. I. FIGURE I M — Extracts from peat mulched plants. U — Extracts from unmulched plants. u Amino-Acids A - Glutamic Acid B - Asparagine C - Proline D - Tyrosine E - Alanine 30 TABLE 71 A relative evaluation of the nitrogen fraction found i n the leaves of unmulched, peat mulched and sawdust mulched plants i n the form of amino-acids Amino-Acids Unmulched Mulched Sawdust Peat Glutamic acid Wt W W Alanine t W w ftt Asparagine W W w t W Proline W t t w tt Tyrosine w *• t Phenylalanine W W tt tt Average to t a l W + f W tt Table 71 indicates that the greatest accumulation of amino-acids was found in the leaves of the unmulched plants, despite the fact that the greatest total amount of nitrogen was present i n the leaves of the mulched plants (table I T " ) . 3 1 Discission of Results The beneficial effects of the peat and sawdust mulches were pronounced* Under f i e l d conditions these benefits have been attributed to factors 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 limiting* It would appear that in seme manner, the mulches in 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 reflected by increased growth of the mulched plants, a higher ash content, and a higher total nitrogen content of the leaves. The pH values of the nutrient media ranged within the limits of pH 5*7 - 6.0 for 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 of pH 4.0 - 5 . 2 is optimum for blueberries, and above or below this pH range, blueberries may bot be expected to survive. The present work does not support this contention, but rather, that blueberry plants may thrive equally well at the higher pH ranges of pH 5 . 7 - 6.0 provided that an adequate nutrient supply is maintained. This i s in agreement with Boiler (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 ratio in the peat than i n the sawdust. The sawdust would then tend to depress available nitrogen 32 i n s o i l . However, in t h i s experiment nitrogen was added in adequate amounts i n a l l oases. The presence of growth hormone substances in the peat and not present, or present in lesser amounts, in the saw-dust i s a possibility, but this was not investigated. Plants mulched with sawdust showed a much greater growth than the control plants, or those to which leachates of peat or saw-dust had been applied. This beneficial effect of sawdust mulch on blueberries agrees with the findings of Savage and Barrow ( 4 5 ) . Plants to which steam.treated leachates were applied grew better than those aupplied ,with untreated leachates. It is possible that the steam treatment softened the c e l l walls of the peat and saw-dust and thus allowed for a more efficient extraction of the water soluble nutrients present in the mulches, and so increased certain nutrient concentrations available to the plant. Further work on the water soluble nutrients of peat and sawdust at various stages of de-composition, treated and untreated with steam, i s needed before a clear picture of the factors contributing to the benefits of steam treatment can be formulated. The leaves of the mulched plants contained a higher percen-tage of total nitrogen but a lower concentration of amino-acids than the leaves of the unmulched plants. This may indicate that the amino-acids in the mulched plants were combining more readily than in the un-mulched plants to form proteins. Six amino-acids were identified. Of these, two, v i z . glutamic acid and alanine are known to play important roles i n protein and carbohydrate metabolism. Glutamic acid i s probably synthesized in plant cellB by a 33 reaction between ammonia, often originating from the reduction of nitrates, and eC-ketoglutarie acid, as follows: COOH COOH (3=0 CHNHg CHgWHj* DPN.Hg *• CH^H^OfDPN COOH COOH <C- Ketoglutaric acid. Glutamic Acid. This reaction i s catalyzed by the enzyme dehydrogenase, with diphosphopyredine mucleotide (DPN) as the coenzyme. This enzyme i s widely and probably universally prosent in plants. Although de-t a i l s of this reaction have been worked out only i n animal tissues (von E i l e r et a l ) , there is l i t t l e doubt that this reaction also occurs in plant tissues. This type of reaction is known as reductive amination. The other amino-acid of great significance found present in the samples was alanine. This acid may have been formed from pyruvic acid by reactions analogous to those stated above. A close interrelation exists between the synthesis of certain amino-acids, organic acid metabolism, and the process of aerobic res-piration. The reversible reactions involving the conversion of •C-ketoglutaric acid to glutamic acid, alanine to pyruvic aoid, and other analogous reactions which may occur can be considered as side reactions of the tricarboxylic acid cycle. There is considerable evidence that some of the amino-acids may arise as a result of transmination reactions in which amino groups 34 are transferred from one kind of a molecule to another. The follow-ing is an example of such reaction: COOH COOH I I C aO transaminase 4- CH3 COOH CHNH COOH glutamic acid Pyruvic acid f 2 COOH COOH CBg Alanine rf-ketoglutaric acid Reactions of this kind are catalyzed by an enzyme of the type called a transaminase. Such enzymes are widely distributed in, higher plants. Due to the above reactions glutamic acid and alanine may be converted respectively to eC-ketoglutaric acid and pyruvic acid and here enter in the tricarboxylic acid cycle in carbohydrate metabolism. Thus i t may be assumed that these two amino-acids were re-acting more rapidly in the mulched plants than in the unmulched plants; and thereby this may account far their 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 of leaves i n plants from the mulched plots. Assuming that the amino-acids, glutamic acid, and alanine transaminate and deaminate to form oC-ketoglutaric atfid and pyruvic 35 acid and thereby enter the tricarboxylic acid cycle, they would cause an increase in the respiration rate and subsequently protein synthesis, and f i n a l l y increase the rate of growth. The better u t i l i z a t i o n of nitrogen i n the metabolic pro-cesses of the blueberry as a result of mulching i s of especial interest and warrants further investigation as to the mechanism involved. 36 CONCLUSIONS From the results obtained, i t may be concluded that blue-berry plants grow at a faster rate when mulched with sawdust or peat than when unmulched. Free amino-acids are used up much faster i n the mulched plants than i n the unmulched ones; resulting in a higher protein content in the mulched plants than i n the unmulched plants. The application of mulches in blueberry culture on mineral soil s can contribute to increased yields. Although only 6 amino-acids were recognized, this does not indicate that there are only 6 free ami no-acids present in 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 to blueberry growing on mineral soils would appear to be a sound cultural practice. 37 SUMMARY Experiments involving the use of Coville blueberry plants were conducted in the f i e l d , greenhouses, and laboratories of the Division of Plant Science at the University of British Columbia to determine the effects of various mulches and their leachates on the growth and certain metabolic responsis of highbush blueberry plants. The results 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 solution. (4) Steam treated sawdust leachate, untreated sawdust leachate, and untreated peat leachate caused an increase in growth but this was not significant. (5) Plants to which the steam treated peat leachate was applied showed a significant increase in growth over the control plants. (6) There was l i t t l e variation i n pH between various treatments throughout the experiment. The pH ranged from 5.7 to 6.0. (7) 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 free amino-acids occurred in the leaves of unmulched plants than in those of the mulched plants. (9) The percentage of oven dried 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 protein in leaves from the mulched plants was higher than in leaves from the unmulched plants. 39 LITERATURE CITED 1. Anderson, J.A. The influence of available nitrogen on the fermentation of cellulose i n the s o i l . S o i l S c i . 21: 115-126, 1926. 2. Aries, R.S. The uses of lig n i n as a s o i l builder. North eastern Wood Ut i l i z a t i o n Council Bulletin 19: 79-122. 1948. 3. Association of O f f i c i a l Agricultural Chemists. O f f i c i a l and tentative methods of analyses. Washington. 4. Awapora, J. Application of paper chromatography to the estima-tion of some free amino-acids in tissue of the rat. Jour. Biol. 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 chrom-atography. Sc. 109: 385, 1949. 6. Bailey, J.S. and J.N. Everson. Further observations on chlorosis of the cultivated 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. Cir. 457. 1945. 8. , and C.A. Doehlert. F e r t i l i z e r and t i l l a g e for blue-berries, New Jersey Agr. Exp. Sta. Bui. 558. 1933. 9. Berry, H.K. and I . Cain. Biochemical individuality. 17. A paper chromatographic technique for determining excretion of amino-acids in. the presence of interfering substances. Arch. Biochem. 34: 179-189. 1949. 10. Blasberg, C.H. Growing blueberries in Oregon. Oregon Agr. Exp. Sta. Pamp. 19. 1948. 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 chrom-atograms. Sci . 109: 608-609. 1948. 13. , Boiling, D. and H.A. Sober. Quantitative separation and determination of small amounts of histidine and tyrosine employing paper chromatography. Federal Proc. 8: 185. 1949. 40 14. Boiler, C.A. Growing blueberries. Ore. Agr. Exp. Sta. B u l l . 499. 1951. 15. Bull, H.B., Hahn, J.W. and V.H. Baptist. F i l t e r paper chrom-atography. Jour. Amer. Chem. Soc. 71: 550-553. 1949. 16. Cain, J.C. Growth and development of blueberry f r u i t . Proc. Amer. Soc. Hort. S c i . 59: 167-173. 1952. 17. . Relationship between iron chlorosis of blueberry and the pH of leaf tissue. Abs. Amer. Soc. Hort. S c i . Sept. 1953. 18. Chendler, F .B . Effect of lime on low bush blueberry. Proc. Amer. Soc. Hort. S c i . 36: 477. 1939. 19. Collison, R.C. Making s o i l s acid for blueberries. New York Agr. Exp. Sta. Quart. Bui. 82: 8. 1942. 20. Consden, R., Gordon, A.H., and A.J.P. Martin. Qualitative analysis of proteins: A partition 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 fields i n New Jersey. New Jersey Agr. Exp. Sta. Cir, 483. 1944. 22. , and J.W. Shive. Nutrition of blueberry i n sand cultures. S o i l S c i . 41: 341-350. 1936. 23. Dunn, S. and J . Sieberlich. Uses of lignin i n Agriculture. 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 quan-titat i v e estimation of amino-acids by paper chromatography. Nature 165: 846-847. 1950. 26. Griggs, B.H. and H.A. Rollins. Effects of soil-management on yield, growth, moisture, and ascorbic acid 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 blue-berry. Pro. S o i l . S c i . Soc. Amer. 9_: 133-141. 1944. 28. Harris, G. Howell. Sawdust as a mulch for strawberries. S c i . Agri. 31: 52-60. 1951 41 29. Johnston, S. Influence of various s o i l s on growth and productivity of highbush blueberry. Mich. Quart. Bui. 24: 307-310. 1942. 30. Johnston, S. Behaviour of highbush and lowbush blueberry selections and their hybrids growing on various soils located at different levels. Mich. Agr. Exp. Sta. Bui. 205. 1948. 31. Johnston, W.A., and I.M. Ware. Effects of different mulches on s o i l acidity. Proc. Amer. Soc. Hort. S c i . 55: 285-288. 1950. 32. Keston, A.S., Udenfriend, S. and M. Levy. Paper chromato-graphy applied to the isotopic derivative methods of analysis. Jour. Amer. Chem. Soc. 69: 31-51. 1947. 33. Kramer, A. and A.L. Schrader. Effects 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 of pH of blue-berry leaves. Plant Phys. 20: 30-36. 1945. 35. Martin, A.J.P. and R. Mittelman. Quantitative micro analysis of amino-acid mixtures on paper partition chromatograms. Biochem. Jour. 43: 353-358. 1948. 36. McHargue, J.S. The role of manganese in plants. Jour. Amer. 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. Stein. Photometric ninhydrin method for use i n chromatography of amino-acids. Jour. B i o l . Chem. 176: 367. 1949. 39. Naftalin, L. Quantitative chromatographic estimation of amino-acids. Nature. 161: 763. 1948. 40. Perlmutter, F. and G.M. Darrow. Effect of s o i l media, photo-period and nitrogenous f e r t i l i z e r on the growth of blue-berry seedlings. Proc. Amer. Soc. Hort. S c i . 40: 341-346. 1942. 41. Poison, A. Quantitative partition chromatography and the com-position of E. c o l i . Biochem. et Biophys. Acta. 2: 575-581. 1948. 42 42. , Moselay, V.M. and R.W.C. Wyckoff. The quantitative chromatography of s i l k hydrolysate. Science 105: 603-604. 1947. 43. Pope, C.C. and M.F. Stevens. Microestimation of amino-nitrogen and i t s application of paper partition chroma-tography. 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 direct photometry. Jour. Amer. Chem. Sec. 71: 4121-412&. 1949. 45. Savage, E.E. and G-.M. Darrow. Responses of blueberries under clean cultivation 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 cul-tured practices on the growth and yield of blueberries, Proc. Amer. Soc. Hort. S c i . 57: 64. 1951. 47. Turk, L.M. The effect of sawdust on plant growth. Mich. Agr. Expt. Sta. Quar. Bui. 26: 10-22. 1948. 48. , and N.L. Partridge. Effects of various mulching materials on orchard s o i l s . S o i l S c i . 64: 111-125. 49. Woiwood, A.J. Micro-estimation of amino-nitrogen and i t s application to paper partition chromatography, Nature 161: 169. 1948. 43 APPENDIX I Analysis of the effect of 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 6 60.92 60.91 60.14 181.97 TREATMENT TOTALS 365.27 365.23 360.33 1090.83 Total S.S. = 60.82s f 60.852 4- ... 60.052 f 60.142 - 1090.832 = 3.29 Between Treatments S.S. r 565.272 * 565.23s f 560.338 - 1090.85s a2.71 6 18 Error S.S. = 3.29-2.71 s .58 Analysis of Variance  S.S.. D.F. M.S. F. Required f Factor 0.05 0.01 Total 3.29 17 Between Treatments 2.71 2 1.355 3.57 3.68 3.36 Error .58 15 .38 ^ . 0 5 2.131 .38 x 6 x 2 = 4.62 44 APPENDIX II Analysis of the effect of sawdust and peat mulches on the total mineral (ash) 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 =• Peat mulched plants TREATMENTS SAMPLES B C G TOTALS 1 1.29 1.53 1.52 4.34 2 1.28 1.51 1.52 4.31 5 1.28 1.54 1.52 4.34 4 1.37 1.50 1.53 4.40 5 1.36 1.55 1.53 4.44 6 1.38 1.50 1.54 4.42 TREATMENT 7.96 9.13 9.16 26.25 TOTALS Total S.S. = 1.293 + 1.282 + 1.28s—1.53s + 1.53s * 1.54s - 26.25 = .17 Between treatments S.S. = 7.96s 4- 9.15s f 9.168 - 26.25s s .16 6 18 Error S.S. = .17 - .16 = .01 Analysis of Variance Required f Factor S.S. D.F. M.S. F a.©5 0.01 Total .17 17 Between Treatments .16 2 .08 114 3.68 6.36 Error .01 15 .0007 L S D . = 2*131 .0007x6x2 . .192 45 APPENDIX III Analysis of the effect of sawdust and peat mulches on the total nitrogen 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 = Peat mulched plants. SAMPLES TREATMENTS B C G TOTALS 1 .4005 .4389 .4340 1.2734 2 .4005 .4382 .4340 1.2727 3 .4006 .4390 .4338 1.2734 4 .4006 .4212 .4300 1.2518 5 .4007 .4211 .4301 1.2519 6 .4007 .4210 .4302 1.2516 TREATMENT TOTALS 2.4033 2.5794 2.5921 7.5748 Total S.S. - .40052 4- .40052 f ••••43012 f .43022 - 7.57482 = .0041 18 Between Treatments S.S. = 2.40332 f 2.57942 f 2.59212 - 7.57482 r .0037 6 18 Error S.S. a .0041 - .0037 Z .0004 Analysis of Variance Required t Factor S.S. D.F. M.S. F .05 .01 Total .0041 17 Between treatments .0037 2 .00185 61.67 3.68 6.36 Error .0004 15 .00003 LSD. 0 5 = 2.131 .0003x6x2 » .12786 


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