Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Biological control of olive green mold in the cultivation of Agaricus bisporus Tautorus, Thomas Edward 1983

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1983_A6_7 T39.pdf [ 8.89MB ]
Metadata
JSON: 831-1.0095833.json
JSON-LD: 831-1.0095833-ld.json
RDF/XML (Pretty): 831-1.0095833-rdf.xml
RDF/JSON: 831-1.0095833-rdf.json
Turtle: 831-1.0095833-turtle.txt
N-Triples: 831-1.0095833-rdf-ntriples.txt
Original Record: 831-1.0095833-source.json
Full Text
831-1.0095833-fulltext.txt
Citation
831-1.0095833.ris

Full Text

BIOLOGICAL CONTROL OF OLIVE CREEN MOLD IN THE CULTIVATION OF AGARICUS BISPORVS by THOMAS EDWARD TAUTORUS B . S c , University of British Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Microbiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1983 (c) Thomas Edward Tautorus, 1983 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y 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 r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f MICROBIOLOGY  The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall V ancouver, Canada V6T 1Y3 Date A p r i l 20 / 1983 DE-6 n / a n A B S T R A C T The Canadian mushroom i n d u s t r y is e x p e r i e n c i n g the same problems as are encountered worldwide - attacks of diseases and pests ( l n g r a t t a , 1980). S u c c e s s f u l methods to control the damaging weed mold -Chaetomium olivaceum (Olive green mold) in mushroom beds are p r e s e n t l y not known. T h i s thesis i n v e s t i g a t i o n attempted to control C. olivaceum by biological means. A thermophilic B a c i l l u s sp (resembling B. coagu-lans - resistant to 0.02% sodium azide, acidophilic) which showed dramatic a c t i v i t y against C. olivaceum on TSY ( T r y p t i c a s e soy agar + 0.1% Yeast extract) agar plates was isolated from commercial mushroom compost (phase I ) . When inoculated into conventional and hydroponic mushroom beds, the B a c i l l u s not only p r o v i d e d a s i g n i f i c a n t degree of protection from C. olivaceum but also increased y i e l d s of Agaricus bisporus. T h i s is the f i r s t isolation of a microorganism i n h i b i t o r y to Oliv e green mold. The B a c i l l u s was shown to produce an extremely potent and stable a n t i b i o t i c (named Chaetomacin) e f f e c t i v e over a wide range of both pH (2-10) and temperature (-15°C to 150°C). Chaetomacin is soluble i n most polar solvents and insoluble in non-polar solvents. T h i s an t i b i o t i c produced at mesophilic temperatures is also active against other B a c i l l u s species and various eukaryotes - but demonstrates no a c t i v i t y against Cram negative organisms or Gram p o s i t i v e c o c c i . F i n a l p u r i f i c a t i o n of Chaetomacin was accomplished t h r o u g h t h i n layer chromatography on Silica gel analytical plates. Amino acid analysis revealed the antibiotic to be a peptide, acidic in nature. Examination of the literature reveals no other previously isolated antibiotics which are identical to Chaetomacin. iii TABLE OF CONTENTS Page ABSTRACT ii LIST OF TABLES viii LIST OF FIGURES ix ACKNOWLEDGEMENTS xi INTRODUCTION 1 LIT ERA T URE REVIEW 4 A. Current Methods of Mushroom Production 4 1. Phase I 4 2. Phase II 13 3. Spawning and Spawn-run 16 4. Casing 20 5. Fruiting . . . . 24 B. Weed Molds (Chaetomium olivaceum) 26 C. Biological Control 32 METHODS AND MATERIALS 37 I. Selection of Thermophiles 37 II. Identification of Thermophiles 42 1. Microscopic Appearance 42 2. Macroscopic Appearance 42 3. Motility « iv Page 4. Anaerobic Growth 43 5. Maximum and Minimum Temperatures of Growth . 44 6. Biochemical Reactions 44 III. Use of Bacillus AOG in Mushroom Cultivation 48 A. Conventional Production of Agaricus bisporus .. 48 B. Hydroponic Production of Agaricus bisporus . . . 50 IV. A n a l y s i s of Inhibitor Produced by Bacillus AOG 54 A. Determination of a pH Change 54 B. Methods of Extracting Inhibitor from Cell-free Extracts of Bacillus AOG 55 i) thermophilic conditions 55 ii) mesophilic conditions 57 iii) evaporation method 58 C. Temperature Stability 59 D. pH Stability 59 E. Solvent Solubility 60 F. Spectrum of Activity of Antibiotic 61 G. Thin-layer Chromatography 61 H. Ultraviolet Spectrum 65 I. Column Chromatography 66 i) Ion-exchange 66 ii) Slephadex LH 20 67 J. Fluorescent Spectrum 68 K. Amino Acid Analysis 68 v. Page RESULTS AND DISCUSSION 69 I. Isolation of Thermophiles Antagonistic Towards Chaetomium olivaceum 69 II. Identification of Isolated Thermophiles 73 III. Cultivation of Agaricus bisporus with the Thermophile Bacillus AOG 83 1. Conventional Methods 83 2. Hydroponic Methods 86 IV. Analysis of Inhibitor Produced by Bacillus AOG 97 A. Effect of pH 97 B. Extraction of Antibiotic 98 i) Thermophilic extraction 98 ii) Mesophilic extraction 99 C. Temperature and pH Stability of Antibiotic 103 D. Solvent Solubility 103 E. Spectrum of Activity 105 F. Thin-layer Chromatography 105 G. Column Chromatography 110 i) Ion-exchange 110 ii) Sephadex LH 20 110 H. Ultraviolet Spectrum 113 I. Fluorescent Spectrum 113 J. Amino Acid Analysis 116 v i . Page CONCLUSIONS 123 LITERA T URE CITED 126 APPENDICES 142 A. Statistical Analysis of Mycelial Diameters 143 B. ; Statistical Analysis of Mushroom Yields 172 vii. LIST OF TABLES Table Page 1. Microorganisms tested, for susceptibility to antibiotic 6 2 2 . Microscopic observations and colony morphology of isolated thermophiles 7 4 3 . Properties of isolated thermophiles 7 6 4 . Summary of properties of known thermophilic Bacillus species 7 8 5 . Extraction of inhibitor from TSY agar discs 1 0 0 6 . Solvent solubility of antibiotic 1 0 4 7 . Solvent systems investigated to determine optimum TLC separation of crude antibiotic 1 0 7 8 . Amino acid analysis of Bands I and II 1 1 7 viii. LIST OF FIGURES Figure P a g e 1. Extensive contamination of mushroom compost bed by Chaetomium olivaceum 30 2. Selection of thermophiles for activity against Chaetomium olivaceum 41 3. Aerobic waste fermenter used for preparation of liquid compost 51 4. Ultraviolet spectra of lignin extracted from wheat-straw and compost 70 5. Thermophiles showing varying degrees of antagonism towards C_. olivaceum on TSY agar 72 6. Gram reaction of Thermophile #10 75 7. Inhibition of Olive green mold on TSY agar by Bacillus AOG 82 8. Mycelial development in standard mushroom compost 84 9. Yield of mushrooms in standard mushroom compost 85 10. Mycelial development in 2% Malt extract 88 11. Poor spawn growth in hydroponic tray with only Olive green mold present , 89 12. Improved mycelial development due to the biological protection of Bacillus AOG against C_. olivaceum 89 ix. Figure Pq.ge 1 3. Yield of mushrooms in 2% Malt extract 90 14. Mycelial development in liquid compost 91 15. Yield of mushrooms in liquid compost 92 16. Fermentation of 2% Malt extract by Bacillus AOG 94 17. Inhibition of C_. olivaceum from cell-free extracts of Bacillus AOG , 102 18. Spectrum of activty of Bacillus AOG against various microorganisms 106 19. Antibiotic elution profile in cation exchange resin 111 20. Ultraviolet absorption spectra of lower (Band I) and upper (Band II) TLC bands 114 21. Fluorescent spectrum of TLC Band I 115 x. ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to Dr. P.M. Townsley, Dept. of Food Science, for his invaluable advice, guidance and enthusiasm throughout the course of this study. He is also thankful to the members of his committee, Dr. J.J.R. Campbell, Dr. A. Ells, and Dr. D. Syeklocha, for their interest in, patience, and review of this thesis. Thanks are also extended to Mr. S. Yee for'his technical assistance and Mr. R. Yada for statistical analysis. The author is extremely grateful to Ms. Cheryl Craig for her encouragement, inspiring criticism, and her endless work in preparing the artwork and manuscript of this thesis. The support of the B. C. Dept. of Agriculture is also gratefully acknowledged. 1. INTRODUCTION The c u l t i v a t i o n of the common white mushroom, Agaricus bisporus Lange {Agaricus brunnescens) has expanded tremendously in recent y e a r s . The B r i t i s h Columbia M i n i s t r y of A g r i c u l t u r e (1981) states that 8820 tons of A. bisporus were produced within th i s p r o v i n c e in 1981 ( r e p r e s e n t i n g approximately a 300 ton increase over 1980). Mushrooms continue to rank second in economic importance as a vegetable crop in B.C. ( t h i r d overall in Canada). Furthermore, B r i t i s h Columbians lead the continent (and p o s s i b l y the world) i n mushroom consumption per c a p i t a , consuming twice that of fellow Canadians and three times as much as U.S. consumers (Anon., 1981; Potter, 1980). It is l i k e l y that the mushroom i n d u s t r y wi l l become of greater importance in the production of enzymes, antitumor compounds, and a n t i b i o t i c s , as well as food and feed (Kurtzman, 1979). However, i r r e s p e c t i v e of p u b l i c demand and food values, the f u t u r e prospects f or continued expansion and mass production l a r g e l y concerns the economics of production methods (Hayes S N a i r , 1975). T r a d i t i o n a l l y accepted as a h o r t i c u l t u r a l c r o p , mushroom production is in p r i n c i p l e a fermentation process (Hayes, 1974; Kurtzman, 1979). P r e s e n t l y mushroom c u l t u r e represents the only major process in biotechnology which s u c c e s s f u l l y converts cell u l o s i c s into useful foods and b y p r o d u c t s . One cyc l e consists of d i s t i n c t procedures: namely su b s t r a t e p r e p a r a t i o n , inoculation, i n c u b a t i o n . 2. growth, and t e r m i n a l d i s i n f e c t i o n ; all of which can be id e n t i f i e d with s tandard laboratory methodology i n microbiology and in the methods employed in the fermentation i n d u s t r y (Hayes, 1974; Hayes & Wright, 1979). The p r i n c i p l e s i n v o l v e d in c u l t i v a t i o n are common to other i n d u s t r i a l processes i n v o l v i n g microorganisms, the guarantee of p u r i t y of c u l t u r e which in t u r n , provides for the guarantee of ed i b i l i t y - is the basis on which the i n d u s t r y was founded. The methods used in this solid-state fermentation have not changed s i g n i f i c a n t l y over the ye a r s . The main sub s t r a t e which supports mushroom growth continues to consist of animal manures, plant materials, chemical f e r t i l i z e r s , and other a g r i c u l t u r a l residues. It must be kept in mind that mushroom compost is a r i c h n u t r i e n t s u b s t r a t e i n which there is an association of competitive microorganisms d i s t i n g u i s h e d by differences in ph y s i c a l requirements, products of metabolism, and various n u t r i t i o n a l requirements (Lambert, 1938). More-over, with the hig h labor input and the pure c u l t u r e e s s e n t i a l i t y , the p r o f i t margin leaves l i t t l e to offset crop e r r o r and f a i l u r e due to diseases and pests. Fungi which grow in competition with the mushroom mycelium are r e f e r r e d to as weed molds and are considered serious disease causing agents (Lambert S A y e r s , 1 953). One such organism is Chaetomium olivaceum more commonly known as Olive green mold. Oliv e green mold is a weed mold which f r e q u e n t l y o c curs a f t e r p a s t e u r i z a t i o n of the compost and severely i n h i b i t s mushroom mycelial development by competing for n u t r i e n t s (Beach, 1937). Consequently, mushroom 3. yields may be completely des t royed . One reason for the invasion of C. olivaceum into mushroom beds is the presence of free ammonia in the compost after the phase II pasteurization (Rettew, 1948). Insufficient oxygen du r ing phase II leads to anaerobic decomposition of the compost. Th i s produces compounds toxic to mushrooms while encouraging colonization of the compost by Ol ive green mold (Nair, 1980). A s of yet , there are no known methods to successfu l ly control this pest. (Vedder , 1978). Economic yields require that a grower use the best techniques pertaining to fungal physiology and disease protect ion. From earl ier research (Huhnke, 1970; Nair & Fahy , 1972; Stanek & Rysava-Zatecka, 1970; Towns ley, 1974), it was suggested that a certain degree of protection from invasion of the mushroom compost by disease causing organisms may be obtained by pr ior fermentation with selected micro-organisms. Huhnke (1970) states that, by inoculating specif ic thermo-phil ic microorganisms into steri l ized compost the cultures are capable of caus ing a selective protection of the substrate against diseases and pests. Hence the objectives of this thesis investigation were to select specif ic thermophil ic microorganisms which would not only support the mushroom, but protect it from damage from Ol ive green mold. Fur thermore, it was hoped that this thermophile(s) could be used in the preparat ion of a hydroponic system uti l iz ing synthet ic substrates ( there-by eliminating the potential hazards of compost) as a growth medium for Agaricus bisporus. If success fu l , the results of this research should facilitate the emergence of the mushroom indus t ry into a level of technology exper ienced in modern industr ia l fermentations. 4. LITERATURE REVIEW A. CURRENT METHODS OF MUSHROOM PRODUCTION U Phase I Commercial mushroom production always starts with the production of a suitable mushroom compost (Hatch & Finger, 1979; Kurtzman, 1979; Royse & Schisler, 1980). The substrate materials usually consist of manure, straw, corn cobs, organic nitrogen, phosphates, gypsum.and other agricultural residues. A wide range of animal manures are used, including chicken, duck, pig, sheep, goat, yak, buffalo, mule, and elephant (Hayes, 1974). Tree bark and municipal refuse (Block, 1964; Chang, 1980b) can also be utilized to a limited extent as substrate as well. There is no standard pattern in compost formulation - it is based mainly on the availability and price of the raw materials and supplements in the particular growing region (Kinrus, 1974). The raw materials must be subjected to a composting process since no way has yet been found to prepare an uncomposted medium capable of large scale production where fast growing contaminants have access to them (Royse & Schisler, 1 980). The nature of the substrate and its preparation, more than all other aspects of growing, dictates the method by which mushrooms are grown. Virtually all of the mushroom compost used in British Columbia is prepared in bulk by the Fraser Valley Mushroom Growers Association (B.C. Ministry of A g r i c , 1980). Composting in its broadest defintion may be defined as "incomplete 5. microbial degradation of organic wastes" (Muller, 1965). Composting is a dynamic bacteriological process in which the organic raw components are progressively broken down and transferred in a series of biological and chemical changes by thermophilic microorganisms to a form which can be utilized readily by the mushroom mycelium (Burden & Peterson, 1972; Huhnke, 1970). The repression and elimination of parasites and pathogenic competitor organisms of the mushroom take place at the same time (Fordyce, 1970). When properly prepared it (compost) supplies all the organic, mineral, and moisture requirements needed for the production of a satisfactory crop. The essential feature of the system is to subject the manure to an aerobic thermophilic fermentation until it is well decomposed. A raw compost will still be an excellent medium for many of the micro-organisms active or present during the phase I and which may be later antagonistic towards the mushroom (Muller, 1965) Modern techniques of composting are modelled on systems devised by Sinden & Hauser (1953). In this method, there are two main phases: an outdoor composting/fermentation (phase I), followed by an indoor controlled pasteurization (phase II). During the initial stages of composting, mesophilic decomposition is quite rapid, causing temp-eratures to rise to the thermophilic region (70-80°C) (Smith, 1969) and stay there for an extended period (3-4d). Easily transformed materials such as sugars and starch (Hatch & Finger, 1979), hemicelluloses, poly-saccharides, and protein are broken down preferentially (Waksman £ Nissen, 1932a,b). As the easily decomposed material is being used, the microorganisms begin to attack the more resistant part of the substrate. 6. such as cellulose and l i g n i n . The compost piles are t u r n e d four times over a period of 8-9d to maintain aerobic conditions within the stack. The d u r a t i o n of time spent on phase I will depend on many local factors -a i r temperature, type of composting s u b s t r a t e s , moisture, and micro-flora present. The l i t e r a t u r e d e s c r i b i n g the p r o g r e s s i v e chemical changes accomp-an y i n g decomposition of organic manures has been e x t e n s i v e l y reviewed ( B u r r o w s , 1951; C e r r i t s et a l , 1965; C r a y et a l , 1971; Kurtzman, 1979; Maeder, 1960; M a t t i n g l y , 1952; Mull e r , 1965; S c h i s l e r , 1980; Schobinger, 1958; Waksman & Cordon, 1939; Waksman et al,1939; Waksman & I y e r , 1932a,b; Waksman & McGrath, 1931; Waksman & N i s s e n , 1931, 1932). C e l l u l o s e , hemicellulose, and solubles all decrease while l i g n i n increases over the course of composting. Since both l i g n i n and insoluble proteins are r e adily available to mushroom mycelium (Waksman & N i s s e n , 1932b) and not so r e a d i l y useable to r i v a l microorganisms, i t seems probable that the b u i l d up of these products at the expense of the more easily decomposed carbohydrates and soluble proteins is an important component in the complex phenomena responsible for the f i n i s h e d compost attaining a balance advantageous for the mushroom mycelium. The objectives of the mushroom grower is to regulate chemical and p h y s i c a l conditions d u r i n g fermentation so that the f i n i s h e d compost will be suitable for mushroom mycelium to predominate over all of the competitive microbial flora present ( F o r d y c e , 1970; Lambert, 1938; Royse & S c h i s l e r , 1980; S c h i s l e r , 1980). The carbohydrates that are easily broken down must be removed so that other f u n g i will be less apt 7. to grow in competition (Vedder, 1978). Hayes (1969) and Fermor et al (1979) state that this can be accomplished by encouraging the growth of large thermophilic microbial populations to u t i l i z e the simple carbohydrates which would otherwise be available to be used by undesirable mesophilic f u n g i . ' Becauseiof the lower mesophilic numbers, less cellulose and hemicellulose are u t i l i z e d by them; consequently there is a net conservation of n u t r i e n t s which support mushroom growth. The u n d e r s t a n d i n g of the components which f u n c t i o n i n composting led to experiments designed to s h i f t the pattern of the microbiological sequence in the composting fermentation i n favor of the organisms which are unable to break down cellulose and hemicellulose. Increased mushroom yi e l d s were obtained from several workers (Hayes, 1969; Hayes & Randle, 1969a,b; Laborde et a l , 1972; Smith, 1974) by the addition of a carb o h y d r a t e supplement at the beginning of phase I. The addition of n u t r i e n t s at phase I is d i r e c t e d towards the feeding of microbial populations in the compost ra t h e r than d i r e c t n u t r i e n t addition for the mush-room. Sucrose supplementation (Hayes, 1969, 1977) favored high populations of b a c t e r i a , at the expense of actinomycetes, with c o r r e s p o n d i n g increases i n the levels of l i g n i n , cellulose, and hemicellulose; a conser-vation of n u t r i e n t s which was associated with increased y i e l d . Furthermore, supplementation p r o b a b l y caused an increase in bacterial biomass (i e . e x t r a -c e l l u l a r polysaccharide capsules) which is a major source of n u t r i e n t s for A. bisporus (Eddy & Jacobs, 1976; Stanek, 1972). San Antonio (1966) concluded that microorganisms may account for e r r a t i c r e s u l t s obtained, when d i f f e r e n t amounts of supplements were added. 8. Bacteria are probably more important to the mushroom grower than to producers of any other crop (F letcher, 1979). T h e y play a signif icant role du r ing the preparat ion of the compost, initiation of fru i tbodies , and may be a major problem because of the diseases they produce. A l l major groups of microorganisms are active du r ing composting, especial ly thermophil ic bacter ia , fung i , and actinomycetes, and ideally each group is dominant at d i f ferent stages. Waksman S Cordon (1939) and Waksman et al (1939b,c) were the f i rst to emphasize the importance in mushroom composting of a mixed microflora of thermophil ic actinomycetes, bacter ia , and fung i . Since then, there have been a great deal of studies concern ing the role of microorganisms in the preparat ion of compost (Hayes, 1969, 1977; O l iver S Guil laumes, 1976; Stanek, 1972) and of the predominant types and succession of each (Chang & Hudson , 1967; Fe rgus , 1964; Fermor et a l , 1979; Fo rdyce , 1970; Hayes, 1969, 1977; Laborde et a l , 1968; Renoux-B londeau, 1959; Stanek, 1967, 1972; Stanek S Zatecka, 1967). Hayes (1969) examined the bacterial populations du r ing phase I and recorded the succession and ecological cyc le of the major types . A t the beginning of composting Bacillus subtilus and a Flavobacterium p r e -dominated, but after the th i rd day B. stearothermophilus and Pseudo-monas sp. became the major types . Thermophi l ic fungi remain active throughout the process and they condition the vegetable matter for lateruutilization by the mushroom (eg. Humicola, Mucor, St i lbel la). He noted that all composts, within narrow limits, are of a given microbiological composition. O l iver & Guillaumes (1976) found that the occurrence of peak 9. populations of bacteria varied in time with the individual composts examined; reaching a maximum population in the first and /or second week. They reported differences in types of bacteria during composting, but similar total populations were reached in the composts they examined. Stanek (1972) noted a similar succession of bacterial types to those reported by Hayes (1969) and all three groups of workers stressed the importance of Bacillus sp., especially during the first week of composting. The thermophilic bacteria and actinomycete populations are well known, but the mesophilic and thermophilic activity of the fungi have not been well documented (Eicker, 1980; Fergus, 1978; Kurtzman, 1979). Cailleux (1973) recorded fifteen genera" of mesophilic fungi occurring In compos^from the beginning of composting to the start of phase II. The possible role of these fungi in compost and their influence on the mushroom are not yet fully understood. Eicker (1980) reported with regards to mesophilic fungi, a clear successional colonization pattern is evident. She concluded that mesophilic fungi (with high cellulolytic activity) play a more important role in composting and substrate colonization than is generally realized. Bels-Koning et al (1962) demonstrated that the thermophilic species of Humicola are important in the composting process. The heat resistance of the thermophilic fungi "have been studied by Fergus (1971). The competition among thermophilic fungi for the available nutrients determine,to some extent,the type of compost produced. Forms such as Rhizopus, Aspergillus, and Craphia are hazardous (Pope et al, 1962). 10. Lambert (1941) demonstrated that compost produced under aerobic conditions at temperatures between 50-60°C was the most beneficial for mushroom cultivation - this suggests a direct relationship with the activity of specific groups of microorganisms. Hayes (1969) and Fordyce (1970) have shown that the early increase and late decreases of temperature during phase I are paralleled by similar changes in numbers of microorganisms. In a study by Stanek (1967a), the exchange of microbial groups in the substrate during fermentation was affected not only by changes in the substrate temperature, but also by change in composition of available nutrients containing nitrogen. Approximately 80% of the total nitrogen remaining after composting is fixed in the humus-lignin fractions (ie. lignin-N complex) (Muller, 1965; Schisler, 1980). The remainder is probably fixed in microbial protein. The mushroom mycelium consumes these complexes and the microbial protein. It is apparent that high bacterial activity during the early days of composting releases complex organic compounds which are then available for mushroom nutrition (Fletcher, 1979). The exact role of bacteria in the nutrition of the crop is not completely clear. In addition to their role in conserving nutrients by reducing fungal competitors, thermophilic bacteria might also serve directly in the nutrition of the mushroom (Fermor et al, 1979). In experiments by Stanek (1968), many thermophilic microorganisms isolated from mushroom compost actively formed vitamins and amino acids. Also it may be the unique nature of the enzymes and proteins which confer the property of heat stability on thermophiles which may be the link 11. between mushroom growth and a heat produc ing fermentation. F u r t h e r -more, the majority of l ipids used by the mushroom from the compost appear to be intracel lular l ipids of thermophil ic microfloras (Schis ler, 1980). T h e fatty acid composition of thermophil ic fungi substantiates this . It has been suggested that protein and acetate units are obtained by the mushroom primari ly from the biomass of thermophiles built up du r ing the phase I stage (Hayes, 1974). A number of thermophiles have been added to compost in efforts to increase the speed of phase I or to increase the y ie ld of mushrooms. Pope et al (1962) added thermophil ic fungi du r i ng the composting process and Renoux-B londeau (1959) added actinomycetes after phase II - both groups reported increases in y ie ld . Stanek & Rysava-Zatecka (1970) added mixed cu l tures of thermophil ic actinomycetes and bacteria to steri l ized compost. T h e y found mushroom cultures were not contaminated by competitive microorganisms and mushroom mycelium grew well in their presence. Furthermore, Huhnke (1970) states that by inoc-ulating specif ic thermophil ic microorganisms into steri l ized compost, the cultures are capable of caus ing ( through product ion of metabolic b y -products , enzymes, etc.) a selective protection of the substrate against diseases and pests. It would appear that there is a great effort to f ind ways to reduce the amount of time required for composting (Fermor et a l , 1979; Kurtzman, 1979). However, most of the rapid composting methods at some time have had diff icult ies with disease and weed fungi (Chanter & Spencer , 1974). It appears that either they can not reproduc ib ly remove the easily 12. metabolized nutrients or that there are some naturally occurring anti-biotics produced during composting, which are not adequately produced in the rapid methods. Many attempts have been made to accelerate the fermentation by careful preparation of ingredients, mechanical breakdown of vegetable matter (Laborde & Delmas, 1969), and carbohydrate supplements (Hayes, 1969). Recent studies in the United States have shown how mushroom production can be greatly speeded up by inoculating fresh compost with 1% compost which was at the height of its activity. Till (1962) demonstrated that A. bisporus did not require a composted substrate for growth, if the spawn-run in non-composted materials was performed aseptically. A. bisporus produced cellulolytic enzymes (Turner, 1977) and should thus be able to break down a suitable substrate unaided by other organisms. However, it is essential for these substrates to be composted before they can be successfully colonized by the mushroom when competing with a natural flora of other microorganisms (Fordyce, 1970; Hayes, 1969). This method has been studied further (Kurtzman, 1979) but presently does not seem to be feasible due to the high costs - particularly for sterilization equipment. Phase I is complete as soon as the raw materials have become pliable, are capable of holding water, the odor of ammonia is sharp, and the dark brown color indicating carmelization and/or browning reactions have occurred (Schisler, 1980). 13. 2. Phase II Phase II is also called pasteurization, sweat-out, or peak-heating. It is, in essence, the second solid state fermentation in the production of Agaricus mushrooms (Kurtzman, 1979). Phase II has two main purposes: final conditioning of compost so that it will be mushroom specific (elimination of ammonia and readily available carbohydrates) and pasteurization to eliminate insects, nematodes, competitive fungi and seeds of higher plants (Burden & Peterson, 1972; Schisler, 1980; Smith, 1969). Mushroom production on pasteurized compost is essentially a continuation of the composting process, in which under such parameters that we may predict the cultivated mushroom to be the strongly dominant organism (Muller, 1965). A reduction in mesophilic fungi and bacteria is also achieved, but it is useless to try to eliminate all the mesophiles in the melice of a mushroom facility. Once the beds or trays are filled, the compost begins to heat through the growth of the microbes and residual nitrogen is ammonified by urea bacteria such as Proteus, Micrococci and Aerobacter (Royse & Schisler, 1980). Steam is initially added to bring the temperature to about 55-60°C. This temperature is held from a few hours up to 2d depending on the practices of the grower (Barret, 1956). Available microbial foods in the compost, at filling (eg. sugars, hemicellulose, starches, lipids), are utilized by the dynamic microbial populations during the phase II. This results in bacterial products at spawning, which are ultimately utilized by the mushroom during spawns-run. The fermentation (phase II) must remove the last of the easily available 14. nutr ients - produc ing a compost which is a selective medium for the mushroom (Eddy S Jacobs, 1976). Schis ler (1980) states that convers ion of ammonia products to microbial protein is best accomplished by thermophil ic bacter ia , actinomycetes (eg. Streptomyces, Thermonopsora) and fungi ( To ru l a , Mucor ) . By so do ing , the ammonia is incorporated into microbial cells and ultimately is available to the mushroom (Sinden S Hauser, 1953). However, the possible product ion of metabolites or compost degradat ive products that improve or reduce ultimate mushroom yield is also theoretical ly possible (Fergus , 1964). Often the compost becomes whitened by the development of thermo-phil ic actinomycetes ( " f i re- fang" ) (Renoux-B londeau, 1959). However, other workers have reported the presence of molds in the compost (Be l s -Kon ing et a l , 1962; Pope et a l , 1962). T h e mold and actinomycete flora of mushroom compost du r ing phase II has been investigated prev ious ly (Renoux-B londeau, 1959; Fergus , 1964; Tend le r & B u r k -holder, 1961). However, much work remains to be done - the identity of the microorganisms still has to be reso lved. Hayes (1977) demon-strated that the actinomycetes, p e r se, are of little importance except to indicate that other essential changes have o c c u r r e d . Attempts to control and develop a specif ic microflora (Pope et a l , 1962; Renoux-B londeau, 1959) .have so far only been prel iminary. Var ious workers (Hayes, 1970, 1977; Hayes & Randle, 1970) fumigated composts with methyl bromide du r ing phase II. Th i s caused a st imul-ation in numbers of bacteria and an enhanced effect on product i v i ty . Th i s suggests a s trong association between bacterial act ivity in the 15. compost and the overall nutrition of the crop. The improved yields were attributed to the destruction of actinomycetes and fungi which utilize valuable cellulosic foods (Hayes, 1969). Further studies on controlling the microflora population in compost will undoubtedly continue. The major problem in peak-heating is that compost is a relatively good insulator; hence, unless there is a considerable heating from the metabolic activities in the compost, it is nearly impossible to heat the compost to the desired temperature (Kurtzman, 1979). In a study by Ross (1976), at temperatures above 55°C and at 40°C or below, the final composts were not selective and supported the growth of competitive fungi as well as the mushroom. Selective compost were produced over the range of 45-55°C. Recent results by Gerrits (1980) also indicated that the temp for maximum mushroom yield may be below 50°C. Furthermore, in pre-liminary work by Ross & Harris (1982), it was found that ammonia dis-appeared rapidly in phase II at much lower temperatures than those conventionally used. Prolonged phase II has been shown to affect adversely the nutrients in the compost, resulting in lower yields (Hatch & Finger, 1979). Once the smell of ammonia is gone, the compost temperature can be dropped to 25°C for spawning. One of the most urgent needs in mush-room cultivation is a simple quantitative measure of decompostion after phase II to supplement the pH value, and the appearance, smell and feel of the compost now used as indicators of its suitability for mushroom mycelium (Lambert, 1938). 16. 3. Spawning and Spawn-run Once the phase II composting is complete, the compost bed is ready for inoculation. It is inoculated with a pure culture of mushroom mycelium on cereal grain (spawn), originally obtained from selected growths from multispore germinations (Hatch & Finger, 1979; Hayes, 1974). Spawn is also an excellent medium for a large number of fungus species (Christianson & Kaufman, 1969; Eicker, 1980) and many of the species recorded in the colonized substrate during investigations are known pathogens of grain spawn (Bitner, 1972; Ivanovich-Biserka, 1972). Except under pure culture conditions, mushroom mycelium must always be considered as competing with other microorganisms (Lambert, 1938). It is essential that the mushroom mycelium develop as soon as possible; otherwise competitive molds will begin to grow and to hamper the development of the mushroom mycelium (Vedder, 1978). Stanek (1972) states that the function of individual kinds and groups of microorganisms, their mutual relations and their influence on the growth of the mushroom mycelia are largely unstudied. Stanek (1974) demonstrated that when mushroom mycelium colonized a compost, the number of bacteria and fungi decreased. At almost the same time as the mesophilic microorganisms begin to assert their influence on the enviro-ment, the thermophilic population declines (Fordyce, 1970) and Agaricus, which is known to produce an antibiotic (Eger, 1962, 1972; Sinden, 1971) becomes dominant. Other mesophilic actinomycete populations are relatively stable. However, Fergus (1971) reports that not all molds are inhibited 17. by Agaricus, or if so, only in very close proximity to the mycelia. Some fungi, unaffected by the antibiotics,may compete for the available substrate and may even kill the mushroom mycelia with their metabolites (Royse 6 Schisler, 1980). Furthermore, it was shown (Stanek, 1974) that in the vicinity of mushroom hyphae - Gram negative bacteria pre-dominated (causing stimulative effects), whose properties were different from bacteria occurring in the compost without mushroom mycelia. It is considered probable that the advantageous interaction between mushroom mycelium and hyphosphere microorganisms enables mushroom mycelium to colonize a compost rapidly after spawning. Examination of the literature reveals that relatively few studies have been conducted to determine if any fungi are growing in the compost with the mushroom mycelia during the spawn-run; and the effects that they may have (eg. toxic products, increased temperature). Also, the effects of these organisms (eg. competition) on the substrate itself and A. bisporus are largely unknown (Eicker, 1980). LaTouche (1949) listed some unidentified fungi of varying or doubtful status in compost. In addition Fordyce (1970) reported finding mesophilic species (unidentified) of the genera Aspergillus, Fusarium, Mucor, and Penicillium in the first two weeks following spawning. Recently, Fergus (1978) isolated and identified 50 species of fungi during mushroom spawn-run. The fungi found at the end of spawn-run included many known "weed-molds" capable of generating metabolic temperatures that can not be tolerated by A. bisporus. Moreover, the thermophilic molds, actinomycetes, and bacteria causing thermogenesis in phase II can also become active after 18. the inoculation of spawn, and can raise the temperature high enough to injure or kill the mycelium (Sinden, 1971). In a later study by Fergus (1982), it was demonstrated that due to the poor heat resistance of most fungi - if fungi are to grow in the cooled compost after spawning, they must be introduced into the compost at the time of spawning or thereafter. Trigiano & Fergus (1979) demonstrated that most of the fungi d u r i n g spawn-run have the capability of utilizing cellulose, starch, and lipids as food sources from the compost (through production of extra-cellular enzymes). There is evidence that many of these fungi may be capable of degrading lignin. Further studies are needed on the enzymatic abilities of compost fungi so their role in mushroom culture could be more clearly understood. Extracellular polysaccharides are secreted in the form of slime layers or capsules on the exterior of bacterial and many fungal cells during growth. Eddy (1976) showed that the selectivity of the mushroom compost for mushroom mycelial growth was closely related to the composition of the slime. Eddy & Jacobs (1976) and Stanek (1972) demonstrated that bacterial polysaccharides were 2-9 times better utilized by the mycelium than glucose alone. Chemically, this substance consisted of glucose, fructose, mannose, uronic acid and nitrogen (0.26%) (Hayes, 1977). It was suggested that other components of the bacterial cell may also have important roles to play and it is interesting to note that A. bisporus can synthesize the necessary enzymes to degrade fungal and bacterial cell walls ( T u r n e r , 1977). The fermented substrate from a Streptomyces sp. and a Pseudomonas sp. 19. were used by Stanek (1972) as a medium to cu l t i v a t e A. bisporus. The aerated s u b s t r a t e from the Streptomyces sp. supported the growth of the A g a r i c u s mycelium, but allowed contamination; on the other hand, the s u b s t r a t e from the Pseudomonas supported l i t t l e growth but d i d not allow contamination. When the two substrates were mixed, contamination di d not occur and increased mycelial growth was evident. Studies by Stanek & Zatecka (1967) and Treschow (1942), have shown that most s t r a i n s of thermophilic cellulose decomposing actinomycetes produced pantothenic and nic o t i n i c a c i d s , b i o t i n , thiamin, and vitamin Bg. These substances stimulated the growth of the mushroom mycelia. The B vitamins were also formed by thermotolerant f u n g i of the genus Humicola. Grabbe (1969) found that humic acids formed b y microorganisms i n compost were a poor source of n u t r i e n t s for mushroom mycelium. However, amino acids produced by various thermophilic actinomycetes (eg. Strep-tomyces thermovulgaris) could be u t i l i z e d by the mushroom as a source of nitrogen (Bohus, 1959; P a r k , 1971). It is quite evident that the exact role of bacteria in the n u t r i t i o n of the crop is not completely clear ( F l e t c h e r , 1979). F u r t h e r research is obviously needed to esta b l i s h the precise c o n t r i b u t i o n of the microbe to the l i f e c y c l e of the c u l t i v a t e d mushroom. Ten to fourteen days after spawning, the spawn will have grown f u l l y t h r o u g h the compost and will appear as a g r e y i s h white growth over the s u r f a c e of the compost (Hayes & Wright, 1979). T h i s is the time to lower the temperature and apply the casing s o i l . 20. 4. Casing The casing layer consists of one inch material with moisture retaining properties which is laid on top of the compost after the spawn has run and is used to help induce fruiting of the mush-room (Dawson, 1977). Neutralized peat is almost exclusively used for casing soil in British Columbia (B.C. Ministry of A g r i c , 1980). It is now known that the casing layer, although a nutritionally poor substrate, supports a bacterial flora which flourishes under the conditions created by the accumulation of volatile metabolites (Hayes, 1974). What is the actual mechanism which induces initiation of fruitbody formation? The various factors which affect the development and growth of fruiting bodies are only partially understood. Couvy (1972) used a two medium technique, and suggested that a transition from rich to poor medium is a requirement for primordium formation (ie. casing). The experiments of Eger (1961, 1963), Hayes (1972) and Hayes et al (1969) demonstrated that an unidentified biological factor was in some way involved in sporophore production. Eger (1961) showed that by sterilizing casing soil., fruiting did not occur in sterile conditions although it did very readily if non-sterile casing was used. Hayes S Randle (1969b) postulate that composts without a casing do not allow a buildup of effective populations of stimulatory bacteria. Other workers (Curto S Favelli, 1972; Eger, 1962; Hayes S Nair, 1975; Hume & Hayes, 1972; O'Donoghue, 1965; Park & Agnihotri; 1969; Urayama, 1965) have also examined the role of bacteria in this-process. Park S Agnihotri (1969) using sterilized, soil showed that a range of soil bacteria (or their 21. filtrates) would promote fruitbody formation. Hayes et al (1969) found that certain bacteria of the genus Pseudomonas, in particular, P. putida were responsible for the initiation of fruitbodies of A. bisporus. Hayes (1974) suggested that P. putida strains might act by producing iron chelating compounds (siderochromes), capable of binding iron from the sequestering conditions of the alkaline casing layer. Previously, Hayes (1972) had shown that a substantial increase in the number of pinheads occurred by applying ferrous salts to the casing soil. The extent of the activity of Pseudomonas putida has also been shown to be related to mushroom productivity; ie. consistently large numbers of primordia are produced when P. putida is grown in association with A. bisporus (Hayes & Nair, 1974; Hume S Hayes, 1972). Nair & Hayes (1974) subsequently demonstrated that bicarbonate ions in the casing influenced the populations of Pseudomonads and these, together with a possible role in the release of bound or chelated iron, resulted in sporophore initiation. On the other hand. Wood (1976) found that Pseudomonas putida, or suspensions of other bacteria had no effect; neither did iron salts or other iron containing compounds on fruitbody formation. Experiments by Long & Jacobs (1974) on axenic fruiting of A. bisporus showed that any compound involved in fruiting initiation must be of low volatility. Their observations indicated that the role of the casing microflora is more likely to be the removal of one or more self inhibitors of fruiting rather than a positive contribution of fruit-inducing substances. Eger (1972) states that both the action of CC» 2 and volatile organic compounds, and the metabolic activity of bacteria are responsible for fruiting. This was substantiated in the studies by Long & Jacobs (1968) and Nair & Hayes (1974). Bacteria that initiate fruiting are able to utilize volatile metabolites of the fungus as sole carbon sources. Ethyl acetate and acetone may be key metabolites (Hayes et al, 1969) as the soil accumulates these volati.les produced by the mushroom. Other volatiles of interest have been quinones, CO, and ethylene (Kurtzman, 1979). Hayes (1972) found that some hemeproteins and EDTA plus ferrous sulphate stimulated the formation of primordia. Hughes (1963) found that sterols and sterol esters accumulated in developing sporophores. Possibly the bacteria which stimulate sporophore formation supply essential steroidal metabolites. Furthermore, it was shown by Ingratta (1980) that high levels of nematodes at the beginning of the crop tended to result in higher yield. It was postulated that a high population at the beginning of the crop assisted in the distribution of the bacteria thought to be necessary for fruitbody formation. Hatch S Finger (1979) maintain that fruiting is due to a combination of effects: 1) starvation of mycelium, 2) removal of mycelial metabolites 3) diffusion barrier for mycelial initials, 4) C 0 2 gradient, and 5) action of microbial activity. If a specific cause of fruitbody formation can be determined, the need for a casing layer might be eliminated, with obvious advantages to the industry (Ingratta, 1980). Studies on specific stimulators may eventually lead to the development of continous culture systems akin to other industrial processes using microorganisms -23. for example, an t i b i o t i c s and brewing; in which pathogens may be more read i l y excluded (Hayes, 1974; Hayes & Nai r , 1975). Su c c e s s f u l establishment of A. bisporus is governed by factors which control the ecology of the two s u b s t r a t e s , used in c u l t i v a t i o n . Temperature and aeration are the main p h y s i c a l v a r i a b l e s . T h i s is ultimately l i n k e d to the a c t i v i t y of microorganisms which generally operate by a f f e c t i n g the a v a i l a b i l i t y of n u t r i e n t s (Hayes & N a i r , 1975). A degree of chemical control is achieved by p r o v i d i n g the c o r r e c t n u t r i e n t s in the r e q u i r e d proportions to obtain the necessary succession of microorganisms. The casi n g layer should therefore be considered as a su b s t r a t e which not only supports the mushroom but also an associated microflora, which p o s s i b l y benefits the mushroom and supports its growth t h r o u g h the important t r a n s i t i o n from mycelial growth to a. f r u i t b o d y (Hayes & Wright, 1979). Attempts in the f u t u r e should take into account the p o s s i b i l i t e s of biological selection of microorganisms essential for sporophore production (Nair et a l , 1974). 24. 5. F r u i t i n g From the developed mycelial aggregates, primordia or pinheads develop, a pr o p o r t i o n of which develop f u r t h e r into the c h a r a c t e r i s t i c f r u i t s (Chang & Hayes, 1978). The mushrooms appear in "breaks" o r flushes and should be ready f o r p i c k i n g about lOd a f t e r the pinheads f i r s t appear ( B u r d e n & Peterson, 1972; Toleman, 1979). Mushrooms are then harvested from the beds or t r a y s for a period which ranges from 28-60d depending on the pract i c e s of the i n d i v i d u a l grower ( I n g r a t t a , 1980). The total h a rvest decreases following the t h i r d f l u s h (Hayes & Wright, 1979; Vedder, 1978) - rel a t i n g to the depletion of nu t r i e n t s and decline of pH ( C e r r i t s , 1965). There is v i r t u a l l y no knowledge of the mechanisms c o n t r o l l i n g the sequential f l u s h i n g of mushrooms (Hayes 6 N a i r , 1975). A f t e r approximately the f i f t h break, mesophilic bacteria and known pathogens of A g a r i c u s are able to colonize the s u b s t r a t e . A t t h i s point, the production of mushrooms becomes uneconomical and the spent compost is d i s c a r d e d . On many la r g e r i n t e n s i v e u n i t s , the disposal of spent or used compost f r e q u e n t l y poses problems. Recent studies have indicated that microorganisms in spent compost have a d i r e c t role in the etiology of mushroom worker's lung disease ( an e x t r i n s i c a l l e r g i c a l v e o l i t i s p o s s i b l y caused by inhalation of spores of thermophilic actinomycetes, mushroom v i r u s e s , or chemicals present i n compost) ( K l e y n S Wetzler, 1981; K l e y n et a l , 1981). Therefore i n c r e a s i n g attention is being g i v e n to systems of r e c y c l i n g / r e p l e n i s h i n g the n u t r i e n t s which have been u t i l i z e d by the mushroom so that they are reused in the succeeding cycle (eg. hydroponics). Urayama (1961) demonstrated that by spraying Bacillus psilocybe, onto mushroom beds, mycelial density and total production of the fruit-bodies of A. bisporus were increased; also initiation of the fruiting was earlier. Curto & Favelli (1972) also found that treatment of A. bisporus with certain microorganisms (eg. Scenedesmus quadricauda) increased mycelial density, reduced time to first picking, and increased yields. Other workers (Park S Agnihotri, 1969; Park, 1970; Renoux-Blondeaux, 1959) also used selected bacteria to increase yield and decrease time to first picking. Park (1970) tried to stimulate the growth of thermophilic actinomycetes by adding industrial waste to the compost and obtained higher yields of mushrooms in doing so. On the other hand, extracts of Penicillium and Aspergillus species were inhibitory to mushroom yields (Stanek, 1959). Despite the considerable control that has now been introduced into cultivation of mushrooms, in order to maximize full growth potential of the crop considerable individual skill and judgement are required in management - of maintaining the culture free from harmful competitors, pests and pathogens. B. WEED MOLDS {Chaetomium olivaceum) Many fungi isolated from mushroom beds act only in the capacity of weeds infesting the compost or casing layer. These organisms are called "weed-molds" and are considered disease-producing agents (Beach, 1937; Lambert S Ayers, 1953). Often, they are known to be serious competitors of Agaricus bisporus for various nutrients, or they inhibit normal growth of the spawn by detrimental changes produced in the conditions of the compost. To date, the molecules involved in this inhibitory process in mushroom composts has not been fully characterized (Chang & Hayes, 1978; Hayes & Nair, 1975). There is currently no effective control for weed molds (Nair, 1980). Sinden (1972) predicts that the number of weed molds known to interferewith the growth of the mushroom in some way, will increase in the coming years as the problem receives more attention. Chaetomium,olivaceum, more commonly known as Olive green mold, is one of the most common weed molds occurring in mushroom compost beds (Beach, 1937). Olive green mold is a croprophilous fungus which inhibits the growth of mushroom mycelium by competition for nutrients (Chang S Hayes, 1978; Eddy & Jacobs, 1976), or through some toxic factors. It has been known to reduce yields and often cause complete crop failure (Lambert S Ayers, 1953; Nair, 1980). As of yet there are no known methods to successfully control this pest (Vedder, 1978). 27. One of the main reasons for the invasion of Chaetomium into mush-room beds is that some ammonia is left in the compost a f t e r the phase II cookout (peak-heat) or is formed anew (B e l s - K o n i n g et a l , 1962; Hayes, 1977; Rettew, 1948; Vedder, 1978). E x c e s s i v e ammonia in the beds may be due to low amounts of gypsum in the compost d u r i n g phase I ( C e r r i t s , 1977); peak-heating for too short or too long a time, or at too h i g h temperature (above 55°C) (Kneebone S Merek, 1959; Lambert S Merek, 1959; Lambert, 1953; Sinden, 1955) and humidity; or by too much compaction and moisture (ie. lack of f r e s h air) ( A t k i n s , 1972; Hayes S Wright, 1979; S c h i s l e r , 1980; Vedder, 1978). F u r t h e r -more, high concentrations of C 0 2 i n the compost seems to promote the germination of spores of O l i v e green mold (Chang & Hayes, 1978). R e s t r i c t i o n of the airflow (causing anaerobic decomposition) produces as of yet unknown compounds hazardous to the mushroom but readily accesible to Chaetomium ( S c h i s l e r , 1980; S i n d e n , 1971). Ex c e s s i v e or great v a r i a t i o n s in temperature (especially d u r i n g phase II) produces unwanted conversions in the compost (ie. proteins are broken down which can be assimilated by Oliv e green mold (Lambert, 1953), and it is quite plausible that anhydrous ammonia will be produced from the h i g h e r nitrogen compounds already formed (Vedder, 1978). Va r i a t i o n s in temperature may occur when the grower attempts to supplement thermogenesis with a r t i f i c i a l heat such as l i v e steam. Too moist a mushroom bed will also cause conversions to be pushed in the wrong d i r e c t i o n , because s u f f i c i e n t amounts of oxygen are unable to penetrate the compost. In other words, when any of these conditions 28. (ie. lack of fresh air, high temperatures, etc.) causes the conversion of ammonia into proteinacious compounds to be unsuccessful, the level of C. olivaceum will be promoted. Insufficient amounts of available carbohydrates in phase II will lead to incomplete conversion of ammonia and amines and their accumulation in the compost (Chang & Hayes, 1978; Hayes & Wright, 1979). Chaetomium has been shown to tolerate as much as seven times the concentration of ammonia as that tolerated by the mushroom (Vedder, 1978). Although it does not always predominate, C. olivaceum has been isolated from all stages of mushroom cultivation including the spawn-run (Eicker, 1980; Fergus, 1978). Phase I composting does not contribute directly to eradication or exclusion of Olive green mold (Sinden, 1971). There are always cool exteriors on which weed molds can be harbored despite thorough mixing during turning. Further-more, in a study by Hayes (1977), suppression of actinomycete and fungal activity (eg. excessively wet mixtures) before the near completion of the maturation stage, greatly affected the ability of A. bisporus to colonize, favoring the development of Chaetomium species. The spores of Olive green mold are widely distributed in nature and extremely heat resistant (Beach, 1937; Chang & Hayes, 1978; Eastwood, 1952; Kneebone & Merek, 1959). The spores are very resistant and may survive inadequate pasteurization and soil treatment. Its growth is readily disseminated and unrestricted (Nair, 1980), and has been known to spread into sterilized soil and onto wooden 29. shelving (Atkins, 1972). It is characterized by spore containing bodies which appear like small, round, olive green to blackish pustules observable on the strands of straw (Figure 1) (Kneebone £ Merek, 1959; Rettew, 1948). The mold gives the manure a typical dank or musty odor (Vedder, 1978). As it (C. olivaceum) develops more or less densely in the compost, the yield of mushrooms will be influenced to a smaller or greater degree. Consequently if Olive green mold appears in large amounts in a localized area of the compost, no mush-rooms will appear at all. Furthermore, spawn growth is retarded or fails altogether (Stanek, 1967). As indicated by Trigiano & Fergus (1979) Chaetomium probably has the ability to produce extracellular enzymes capable of degrading insoluble, organic compounds such as cellulose, starch, lipids, and lignin (factors which aid in its competition). In a study by Chahal et al (1975) , Chaetomium species demonstrated the highest carboxymethylcellulase activity and highest degradation of wheat straw when compared to a wide variety of other fungi (all isolated from mushroom compost). Degradation of wheat straw was doubled when it was delignified. C. olivaceum is capable of growth over a wide variety of pH (Beach, 1937). Its optimum is pH 6.8, but it grows well at pH 8.0, where the growth of spawn is often inhibited. Olive green mold grows most readily on slightly acid materials but it adapts quite readily to very alkaline manure. Beach (1937) has shown that mushroom mycelium was unable to compete with Olive green mold at any point of the pH scale when flasks of manure were inoculated with both fungi. Figure 1. Extensive contamination of mushroom compost bed by Chaetomium olivaceum. 31. Olive green mold is also found on beds which have been supplemented with n u t r i e n t s a f t e r phase II (Sinden & S c h i s l e r , 1962) or d u r i n g composting (Hayes, 1977; Vedder, 1978). Chaetomium seems to be the limiting factor (in the amount of supplement added) because it competes for the supplement with the mushroom mycelium - with the slow growing mycelium losing out. Chaetomium is also a serious pathogen of cotton, soybean and sun-flower crops (Kanwar et a l , 1979; Nik, 1980). It causes biological damage to wool f i b e r s (Sankov et a l , 1972), and deteriorates s y n t h e t i c resins such as urethane r u b b e r (Takeyoshi et a l , 1971). Chaetomium is c u r r e n t l y known to produce a wide v a r i e t y of d i f f e r e n t toxic metabolites (ie. mycotoxins) (Brewer et a l , 1970; Brewer & T a y l o r , 1978; Sekita et a l , 1981) such as sterigmatocystin (possible p r e c u r s o r to a f l a t o x i n s ) , oosporein, c o c h l i o d i n o l , chaetomin, and the chaeto-globosins. The la t t e r two products have been the responsible agents in several mycotoxicoses. T h i s organism (Chaetomium) has also been implicated in at least one patient death in the United States (personal communication; Dr. M.G. R i n a l d i , Dept. of Microbiology, Montana State U n i v e r s i t y ) .* C. BIOLOGICAL CONTROL Garrett (1965) defines biological control as "any conditions under which, or practice whereby, surivival or activity of a pathogen is reduced through the agency of any other living organism (except man himself), with the result that there is a reduction in incidence of the disease caused by the pathogen". In its widest sense, the definition of biological control can be expanded to include integrated control where chemicals and living organisms can be used successfully in conjunction to control pests.(Hudson, 1972). Microbiological control of plant diseases can be achieved directly through inoculation, or indirectly by changing the conditions prevailing in the plant's environment, and thus the microbiological equilibrium of its ecosystem, or by combination of both systems (Henis & Chet,:1975). Moreover, the inordinately high cost of developing chemicals to control pathogens and the lack of resistance of crop plants to may diseases, has attracted the attention of many scientists and, recently, venture capital companies which see a profitable future (Scroth & Hancock, 1981). A common feature of microbial agents, when compared to chemicals is the widespread resistance they encounter from the receiving biotic environment. Breaking or escaping this resistance is a main condition for the success of biological control (Gindrat, 1979). Except in a few cases, biological control of plant diseases with antagonistic microorganisms is still restricted to experimental work, despite the large amount of published data and reviews on this subject 33. ( B a k e r , 1968; Baker £ Cook, 1974; Baker £ S n y d e r , 1965; G a r r e t t , 1955, 1965; Hussey, 1969; Henis £ Chet, 1975). The extreme d i f f i c u l t y of is o l a t i n g , c o r r e l a t i n g , and unde r s t a n d i n g the many factors that influence microbial a c t i v i t e s around root systems and on plant surfaces has impeded the development of biological control practices of commercial benefit ( G a r r e t t , 1955; S c r o t h £ Hancock, 1981). Thus, despite the many decades of res e a r c h , there are only two cases where a biological control agent has been r e g i s t e r e d for use by a government agency and is commercially used in North America (biological c ontrol of insects is excepted). The probable mechanisms of biological control are that of a l i v i n g organism acting d i r e c t l y on the pathogen (antagonism) or thr o u g h the intermediate agency of the host ( B a k e r , 1968). The two main categories of antagonism are antibiosis and competition ( P a r k , 1960). A n t i b i o s i s (Scroth £ Hancock, 1981) is defined as an interaction between organisms whereby a metabolic agent produced by one organism has a harmful effect on the other. Usually this d e f i n i t i o n excludes common metabolic p r o d u c t s , such as c a r b o x y l i c acids, ethanol, and C 0 2 . T h i s topic has also been reviewed ra t h e r e x t e n s i v e l y in the l i t e r a t u r e ( B r i a n , 1957; G a r r e t t , 1956; Jackson, 1965). Production of anti b i o t i c s on growth media is common among soil f u n g i , actinomycetes and b a c i l l i . How-ever, the func t i o n of antibiosis in biological c o n t r o l , t h e i r formation and ecological s i g n i f i c a n c e in common soil have induced a considerable debate ( B a k e r £ Cook, 1974; Jackson, 1965). 34. Competition refers to the interaction of two organisms striving for the same thing; for example, space, nutrients (Park, 1960; Waksman, 1952) - in other words, active demand in excess of immediate supply of materials or conditions. However, although competition for oxygen and minerals is well known, its role in biological control of plant pathogens is most limited to N 2 (Scroth & Hancock, 1981), which is one of the chief limiting factors in soil. The biological control of crown gall caused by Agrobacterium tumefaciens is the outstanding example of an antagonist that has effectively and economically controlled a major plant disease, has been commercialized and is currently being used in agriculture (Scroth & Hancock, 1981). The application of spores from the fungus Peniophora gigantea to control infection by Fomes annosus in pine stumps is the other case of a biological control that has been effective, widely tested, and registered for use by the government (Risbeth, 1978). The commercial production of the mushroom, Agaricus bisporus lends itself as an extremely feasible system to apply biological methods of control (Hussey, 1969; Nair & Fahy, 1972; DeTrogoff & Ricard, 1976). Loudon (1 850) first suggested that "a toad kept in a mushroom house will eat the vermin, snails, and slugs mentioned, and also worms, and ants and other insects". This interesting statement from an early mushroom grower is an example of the control of pests by biological methods - an approach that is only now, over a century later being considered as potentially valuable in modern cultivation processes (Hayes & Nair, 1975). 35. The concept of biological control was first utilized in mushroom cultivation for control against insect pests. Hussey (1969, 1972), drew attention to the possibility of using nematodes [Bradynema sp and Tetradonema sp) for control of sciarids and phorids. In another study by Hudson (1972), it was demonstrated that nematodes (eg. Tetradonema plicans) could indeed be potentially effective control agents for the insect pests of mushroom crops. However more work is necessary to exploit this possibility and make it commercially feasible. At present there is not a successful method for mass-production of the nematodes (Richardson, 1981). It would appear that this and similar systems of control could be major methods in controlling the insect pests of mush-room crops in the very near future. Pseudomonas tolaasii, the cause of brown blotch in mushrooms, is omnipresent in casing soils and is spread in watering. All previous attempts to control brown blotch effectively have failed (Nair, 1974; Nair S Fahy, 1972). Nair & Fahy (1972) have investigated the possibility of biological control of this disease. Their experiments demonstrated that an effective biological control against P. tolaasii is to use P. multivorais, P. fluorescens, or E. aerogenes added as a peat culture to the casing soil after it is applied to the compost (mech-anism is perhaps by competition for nutrients). Commercial trials with peat inoculants of the bacterial antagonists resulted in successful biological control of the disease, and in some trials increased yields of 8-16% were obtained (Nair S Fahy, 1976). 36. Verticillium malthousei (causing dry bubble in mushrooms) is probably the most widespread and destructive pathogen of mushrooms. It spreads rapidly through the beds. DeTrogoff & Picard (1976) have shown that spraying Trichoderma propagules on casing soil at the rate of 100 x IO6/ I was an effective control of Verticillium. In a study by Tovmsley (1974), it was demonstrated that thermophilic fermentation of spawn grain with the pure culture of a thermophile prior to inoculation with a pathogenic Penicillium conferred varying degrees of resistance to the grain against the mold. It was concluded that a degree of protection from invasion of mushroom composts or spawns by disease causing organisms may be obtained by prior fermentation with selected thermophiles. In a study by Han et al (1974), development of Mycogne perniciosa Magn. (wet bubble) was suppressed by unidentified organisms in the casing soil. Infected sporophores occurred when the casing soil was inoculated on the surface with a spore suspension of M. perniciosa but not when the inoculum was applied to the surface of the mushroom grain spawn or the middle of the casing soil. One of the problems of the mushroom industry at the present time is that of toxic chemical treatments and resistance shown by the pest pathogens, so that it may be advantageous to use biological control in conjunction with lower chemical doses in an integrated control programme (Hussey, 1969). There is no doubt that a correctly conducted biological method can achieve more certain and predictable results than that attainable through normal pesticide usage. 37. METHODS AND MATE R I A L S C u l t u r e s of Agaricus bisporus were received from the F r a s e r Valley Mushroom Growers Co-op (Langley, B.C.) and maintained on Potato dextrose agar (Difco) at 25°C and 3°C. Cu l t u r e s of Chaetomium olivaceum were received from Dr. L.C. S c h i s l e r , Dept. of Plant Pathology, Pennsylvania State U n i v e r s i t y ; C. olivaceum was also maintained on Potato dextrose agar at 25°C and 3°C. I_. S E L E CTION OF THERMOPHILES Representative (40g) samples of commercial compost which had been passed t h r o u g h the i n i t i a l s t a n d a r d mushroom composting stage (phase I) p r i o r to d e l i v e r y to the grower for pasteuriz a t i o n (phase II) were received on a weekly basis from the F r a s e r Valley Mushroom Growers Compost D i v i s i o n . These samples were immediately incubated upon receipt for two days at 55°C under aerobic conditions and high humidity ( F i s h e r Isotemp i n c u b a t o r ) . T h i s incubation p r o v i d e d a controlled d u p l i c a t i o n of the fin a l thermophilic treatment given by the mushroom grower before inoculation with mushroom spawn (Ross & H a r r i s , 1982). For bacteriological a n a l y s i s , lOg of the thermophilic compost was mixed with 90ml of s t e r i l e d i s t i l l e d water and the sample kneaded thoroughly in order to place s u f f i c i e n t microflora into suspension. T h e r e a f t e r , tenfold 38. dilutions using sterile distilled water were prepared to a 10 dilution. -2 -7 Aliquots of 0.1ml from the 10 to 10 dilutions were spread plated in duplicate on TSY agar (Trypticase soy agar (BBL) + 0.4% Yeast extract (Difco) ) and incubated at 55°C for l6-20h. a) Initial screening method for thermophiles antagonistic towards C. olivaceum: Following incubation of plates from above, morphologically different thermophilic colonies were isolated and transferred to new TSY plates for purification. After incubation at 55°C, cultures were transferred to TSY agar slants, incubated until good growth was evident and then refrigerated at 3°C. This procedure was carried out until 45 thermophiles were isolated. To determine if the isolated thermophiles would inhibit Olive green mold (and support A. bisporus) on a synthetic compost medium (see below for components) : cultures were first incubated in 25ml of TSY broth for 48h at 55°C in a shaker water bath (Blue M, Illinois). Following incubation, 2.0ml of the culture broth was used to inoculate the synthetic compost media (4 plates / thermophile). Four control plates containing no thermophile (ie. 2.0ml of TSY broth only) were also prepared. Plates were then incubated at 55°C for 48h under high humidity. After plates had been cooled to room temperature, either a 10mm square agar slab of C. olivaceum or A. bisporus was then placed on top of each plate and incubated at 25°C. Plates were examined on a daily basis for inhibition of C. olivaceum and/or growth of A. bisporus. 39. Because of the poor r e s u l t s associated with t h i s method (see Results S Discussion section) a r e v i s e d procedure was designed (see (b) ). S y n t h e t i c compost medium The s y n t h e t i c compost medium was composed of: C a C 0 3 1. Og ( N H 4 ) 2 H P 0 4 0.7g M g S 0 4 0.2g KCI O.lg F e S 0 4 • 7 H 2 0 lOmg d i s t i l l e d water 1000ml (fi n a l pH 7.0) T h i s n u t r i e n t s solution was then s t e r i l i z e d u s i n g steam for I5min at 15psi. Fifteen ml of the s t e r i l e n u t r i e n t solution was added to 5.5g of s t e r i l e ball-milled wheat straw (in a glass p e t r i dish) and called s y n t h e t i c compost medium. The n u t r i e n t was c a r e f u l l y added at a level just s u f f i c i e n t to evenly moisten the v e r y d r y straw. 40. Lignin analysis of ball-milled wheat straw: i) Lignin content: the lignin content of the ball-milled wheat straw was determined by the acetyl bromide method of Johnson et al (1961). The lignin content of a standard mushroom compost (previously ball-milled) was also determined for comparison. All experiments were conducted in duplicate. ii) U..V. spectra: following extraction of the ball-milled wheat straw and compost (50g) with 100% methanol (100ml), samples were evaporated on a Rotavapor RII6. The concentrated residues (approx. 10ml) were then scanned through the ultraviolet region on a Canlab Unicam SP. 800B UV Spectrophotometer and spectra recorded. b) Revised thermophile selection procedure: Following incubation of the plates from stage I (ie. diluted compost samples) and the appearance of colonies, all TSY plates were sprayed with a spore suspension of C. olivaceum (in sterile distilled water) (Figure 2). The treated plates were then incubated at 25°C and examined daily for the presence of zones of fungal.inhibition. Thermophilic colonies showing antagonism towards C. olivaceum were further purified on TSY agar and later stored on TSY agar slants at 3°C. The 45 previously isolated colonies (from Section a) were also tested in this way. 4 0 g p h a s e 1 c o m p o s t 4 B h ; 5 5 ° C I O g + S O m l s . d . w . * - a - 7 s e r i a l d i l u t i o n I O t o O . I m l s p r e a d p l a t e o n T S Y a g a r 1 6 - S O h ; 5 5 ' s p r a y w i t h s u s p e n s i o n o f C o l i v a c e u m 2 5 ° C e x a m i n e d a i l y f o r z o n e s o f i n h i b i t i o n Figure 2. Selection of thermophiles for activity against Chaetomium olivaceum. II. I D E N TIFICATION OF THERMOPHILES 1_. Microscopic appearance a) Gram s t a i n : Young c u l t u r e s of the thermophiles p r e v i o u s l y grown on TSY agar (12-20h) were Gram stained and observed for size (micrometer) and shape of c e l l s , morphology and p a r t i c u l a r g r o u p i n g s , and presence/location of spores ( u s i n g oil immersion microscopy). b) Spore s t a i n : C u l t u r e s were stained according to the method of Dorner (Doetsch, 1981) and observed for the location and nature of sporebodies (any swelling of the sporangia was also noted). Spores were observed to stain-red against a colorless bacterial c e l l . 2. Macroscopic appearance Thermophiles were observed for colony morp-hology such as s i z e , shape, margin, elevation, pigmentation, etc., on TSY and PDA agars. 43. 3. Motility The motility of young cultures of each thermophile was determined by: a) direct microscopic observation from wet mounts of the organisms (Smibert & Krieg, 1981). b) use of semisolid media (0.7% agar + TSY broth) ;cultures were stabbed to one half the depth of the tube and incubated at 55°C. Motility was indicated by migration of cells through the surrounding medium (Krieg & Ger-hardt, 1981). 4. Anaerobic growth To determine if cultures could grow under anaerobic (obligate or facultative) conditions: a) thermophiles were stabbed to the bottom of a sloppy agar medium (0.7% agar + TSY broth) in duplicate and incubated at 55°C for 5d. Growth on the surface of the agar (aerobic) and along the length of the stab (anaerobic) was recorded by visual obser-vation. b) to confirm a), cultures were streaked onto TSY agar plates in duplicate and incubated in anaerobic jars at 55°C. Anaerobic jars were used with a Gaspak (BBL Microbiology Systems) and catalyst. 44. 5. Maximum and minimum temperatures of growth C u l t u r e s were inoculated into T S Y broth and incubated in duplicate at: a) for maximum temperature determination - 55, 60, 65, 70, and 75°C i n a waterbath (Blue M, I l l i n o i s ) . Growth of the c u l t u r e s were determined a f t e r 3d (Gordon et a l , 1973) by the presence or absence of t u r b i d i t y . D u r i n g i n c u b a t i o n , the water level of the bath was c a r e f u l l y maintained. determination - 15, 25, 35, 45, and 55°C. Fifteen c entigrade was the lowest temperature that could be maintained acc u r a t e l y . Growth was recorded a f t e r 5d at temperatures between 35-55°C and af t e r 21d at 15°C (Gordon et a l , 1973) . 6. Biochemical reactions b) for minimum temperature The following biochemical tests were performed on the thermophiles: a) Catalase te s t : (Smibert & K r i e g , 1981). b) Methyl red tes t : (Smibert & K r i e g , 1981). c) H y d r o l y s i s of urea: (Smibert & K r i e g , 1981). d) H y d r o l y s i s of s t a r c h : (Gordon et a l , 1973). e) Production of indole: (Smibert & K r i e g , 1981). 45. f) Voges-Proskauer: As the formation of acetylmethylcarbinol (acetoin) is one of the most reliable and useful characters in separating Bacillus species (Gordon et al, 1973), two methods for its detection were used: i) method of Smibert & Krieg (1981), utilizing a standard MR-VP broth (BBL). ii) method of Gordon et al (1973), utilizing a revised VP medium. containing 0, 5, 7, and 10% ( /v) sodium chloride were inoculated with cultures previously grown in nutrient broth. Tubes were incubated at 55°C and growth irr the various concentrations of NaCl were recorded at 7 and I4d (Gordon et al, 1973). g) Growth in NaCl: Tubes of nutrient broth h) Growth on Mannitol salt agar: (Smibert & Krieg, 1981). i) Resistance to lysozyme: (Gordon et al, 1973). J) Utilization of sodium citrate: (Gordon et al, 1973). k) Utilization of sodium propionate: (Gordon et al, 1973). I) Reduction of nitrate to nitrite: (Gordon et al, 1973). m) Deamination of phenylalanine: (Gordon et al, 1973). 46. n) Decomposition of t y r o s i n e : (Cordon et a l , 1973). o) Growth at pH 5.7: TSY b r o t h was prepared according to the manufacturer and the pH adjusted to 5.7 with IN HC1. Media was f i l t e r s t e r i l i z e d (0.45 um millipore f i l t e r ) and aseptically d i s t r i b u t e d into s t e r i l e test tubes. C u l t u r e s were inoculated i n duplicate and incubated at 55°C. Growth was recorded at 7 and I4d of incubation. p) Growth in sodium azide: Tubes of azide dextrose b r o t h were prepared by two methods; i) a c cording to the d i r e c t i o n s of Gordon et al (1973). ii) azide dextrose b r o t h ( F i s h e r Gram-Pac, P i t t s b u r g h ) , prepared according to manufacturer's d i r e c t i o n s . Both methods res u l t in a f i n a l concentration of 0.02% azide in each tube. C u l t u r e s were inoculated i n duplicate into both types of medium and incubated at 55°C. Growth was observed a f t e r 7 and I4d of incubation. q) A c i d from c a r b o h y d r a t e s : 10% aqueous solutions of D (-K-)- glucose (Amachem), L (+)- arabinose, D (+)- x y l o s e , and D ( - ) -mannitol were f i l t e r s t e r i l i z e d (0.45um millipore) and aseptically added to phenol red b r o t h base (Difco) to y i e l d a fin a l concentration of 1% sugar in each tube. C u l t u r e s were inoculated into sugars in duplicate and incubated at 55°C. A c i d p roduction was recorded at 2, 7, and I4d. 47. r) Production of dihydroxyacetone: (Cordon et al, 1973). s) Milk reactions: i) Litmus milk; Tubes of litmus milk (Difco) were prepared according to manufacturer's directions and 10ml aliquots were autoclaved for I5min at I5psi. Cultures were inoculated in triplicate and incubated for 7 and I4d at 55°C (Gordon et al, 1973). Tubes were observed for color (acid/alkaline), reduction of indicator (milk appears white due to discoloration of litmus), formation and type of curd (hard or soft), gas and digestion of casein (proteolysis - milk becomes translucent due to hydrolysis of casein). ii) Decomposition of casein; Bacto-Skim milk powder (10g) in 100ml of distilled water, and 2g of agar in 100ml of distilled water were autoclaved (separately) for 15min at 15psi. After cooling to approximatley 45°C, they were mixed together and poured into sterile plastic petri dishes. After allowing plates to dry for 3d, cultures were streaked once across a plate (in duplicate) and incubated at 55°C for 7 and 14d. After the incubation periods, clearing around and underneath the growth indicated casein decomposition (Gordon et al, 1973). 48. M L USE OF B A C I L L U S AOG IN MUSHROOM C U L T I V A T I O N A. Conventional Production of Agaricus bisporus 500g of standard phase II mushroom compost was spawned with 9.0g of Agaricus bisporus spawn g r a i n s (obtained from F r a s e r V a l l e y Mushroom Growers Association) and placed in pl a s t i c growing t r a y s (8 x 15 x 23cm). Elemental analysis of the compost used (Canadian Microanalytical L aboratories, Vancouver) demonstrated it to contain 46.7% Carbon and 2.14% Nitrogen (avg. of 3-50g samples). T h i s spawned compost was then subjected to various treatments: a) control - no f u r t h e r addition to the compost bed; b) 1.00ml of a 10^/ml thermophilic B a c i l l u s AOG c u l t u r e grown in T S Y broth for 96h at 55°C (New B r u n s w i c k Psychrotherm shaker) was added; c) 100ml of a 10^/ml thermophilic Bac i l l u s AOG c u l t u r e grown in T S Y b r o t h for 96h at 55°C was added; followed by s p r a y i n g 2.0ml of a Chaetomium olivaceum spore suspension on top of the bed ; or, d) 2.0ml of a C. olivaceum spore suspension sprayed on top of the bed only. 49. All experiments were conducted in triplicate (except d , which was done in duplicate). The growing trays were then incubated at 25°C for 12d to promote spawn growth. During this incubation, the mycelial diameters of the spawn in each tray were measured on a daily basis (for 7 days) as an indication of the mycelial developments under the various treatments (Brancato & Colding, 1953; Fermor, 1982). After approximatley I week, the spawn spread throughout the bed in most trays and accurate measurements could no longer be recorded. After 12 days, each of the various trays was cased with 2.5cm of a sterile peat/sand/ground lime-stone (Dolomite) mixture in a ratio of 5:5:1 respectively and placed in a 16°C incubator to promote pinhead formation. Air was pumped in at 550 ml/min to keep CC»2 levels at a minimum (ie. C 0 2 is inhibitory to fruitbody development at levels above 0.5%; Long S Jacobs, 1968). Relative humidity was maintained at 80%. As the mushroom-fruitbodies appeared (harvested just prior to opening of gills) yields (expressed in grams freshweight) and date of picking were determined over a I month period. 50. B_. Hydroponic Production of Agaricus bisporus w 2% ( /v) Malt e x t r a c t ( D i f c o ) , containing 2% CaCC» 3 and 0.4% Yeast e x t r a c t (pH 7.0) or a l i q u i d compost solution (see below) were chosen as the l i q u i d n u t r i e n t s for mushroom hydroponic c u l t u r e . L i q u i d compost: L i q u i d compost was prepared by adding 5L of d i s t i l l e d water to Ikg wet weight of mushroom compost. T h r e e lOOg samples of the compost were oven d r i e d at 80°C for 48h to determine total solids present. The mixture was agitated v i g o r o u s l y (584rpm) in a thermophilic waste fermenter' ( F i g u r e 3) with a i r being pumped i n at 4800 ml/min to maintain aerobic conditions. The compost ex t r a c t was then collected by f i l t r a t i o n t h r o u g h cheesecloth and s t e r i l i z e d u s i n g steam at 15psi for I5min. The r e s u l t i n g s u b s t r a t e was called l i q u i d compost. Final pH of t h i s medium was 7.2. To thi s l i q u i d compost and to the 2% Malt e x t r a c t was inoculated the Bac i l l u s AOG c u l t u r e . These l i q u i d s u b s trates were then incubated at 55°C 4 for 96h to y i e l d a f i n a l concentration of 10 cells/ml (determined by plate count on TSY agar) . D u r i n g the above 96h incu b a t i o n , 2ml samples were asepti c a l l y removed on a d a i l y basis from the 2% Malt e x t r a c t medium for total c a r b o h y d r a t e analysis (see below). Figure 3. Aerobic waste fermenter used for preparation of liquid compost. 52. The experimental treatments were similar to the solid compost plan previously described (see Section.A). Either 500ml of a 10 7ml Bacillus AOG culture in liquid compost or in 2% Malt extract was added to 180g of sterile vermiculite (inert carrier material no less than 2.0mm in size) within a plastic tray. To the bottom (centre) of each tray was placed a sterile glass wool plug approximately 20cm long to help ensure aerobic conditions within the bed. Following spawning (9.0g of spawn grains/ tray) , representative trays with or without the Bacillus were sprayed with 2.0ml of a C. olivaceum spore suspension and incubated at 25°C for 12d. The rate of mycelial development was determined on a daily basis for each tray during this incubation (for 7d). All trays were then cased with 2.5cm of a sterile peat/sand/ground limestone mixture (5:5:1 respec-tively) and maintained at 16°C for fruitbody formation. Yields of mushrooms and day of picking were recorded during a 30d cropping period. All hydroponic experiments were conducted in triplicate. 53. Carbohydrate analysis: Total carbohydrate analysis of the 2% Malt extract medium was determined by the phenol-sulphuric acid method of Dubois et al (1956). Tubes were read at 490um on a Beckman Model DB Spectro-photometer. All tests were conducted in triplicate. Statistical analysis: The curves obtained from mycelial diameter data were analyzed for significant differences in slope and level. Analysis of variance and Student Newman-Keul's multiple range test were performed on data for mushroom yields. 54. IV. ANALYSIS OF INHIBITOR PRODUCED BY BACILLUS AOG A. Determination of a pH Change a) Bacillus AOG was streaked once across 6 TSY agar plates and incubated at 55°C for 16-20T). Following incubation, plates were sprayed with a spore suspension of C. olivaceum and incubated for 4d at 25°C (ie. until the zone of inhibition was fully developed on each plate^. The pH of each plate was determined throughout the medium with a Fisher Combination Flat-Surface-Polymer Body Electrode. The pH of 4 un-inoculated (control) TSY plates was also measured. b) 10ml of TSY broth were placed in 18 x 150mm test tubes and sterilzed for 15min at I5psi. The pH of each tube was aseptically adjusted between 2-10 with either IN HC1 or 3N NaOH. C. olivaceum was inoculated into all tubes (conducted in duplicate) and incubated at 25°C for 7d. Evidence of growth (of C. olivaceum) was indicated by lack or presence of turbidity after 7d. 55. B_. Methods to Extract Inhibitor from Cell-free Extracts of  Bacillus AOG i) Thermophilic conditions: a) TSY broth: Bacillus AOG was inoculated into 100ml of TSY broth (in duplicate) and incubated for 2-5d at 55°C in a shaker waterbath (Blue M, Illinois). Following incubation, cultures were centrifuged in a SS-34 rotor for 25min at 15,000rpm (27,000 x G ) , filtered through a 0.22um millipore, and the filtrate then freeze-dried for concentration. The resulting compound was reconstituted in either 1ml of 50% ethanol or 1ml of cold sterile distilled water. Samples (0.1ml) were then soaked onto filter paper discs and placed onto TSY agar plates. Plates were sprayed with a C. olivaceum spore suspension and incubated at 25°C for 5d (examined daily for the presence of zones of inhibition). b) Soybean meal: Bacillus AOG was also grown in 100ml of soybean meal (soybean meal, 4g; C a C 0 3 , 0.5g; starch, 0.5g; distilled water, 1000ml) for 3, 4, 5, 7, and 8d periods at 55°C (in duplicate) and harvested as above. Prior to harvesting, cultures were Gram stained and examined for spore formation. After freeze-drying, cultures were reconstituted in cold sterile distilled water and extracted with n-butanol. The n-butanol extract was then soaked (0.1ml) onto filter paper discs and placed onto TSY agar plates. Plates were sprayed with a spore suspension of C. olivaceum, incubated for 5d at 25°C and examined daily 0for zones of inhibition. 56. c) TSY agar plates: Experiments were conducted to determine if the antibiotic compound could be extracted/ leached out of TSY agar. Bacillus AOG was streaked once across a TSY agar plate and incubated at 55°C for 20h. Following incubation, plates were sprayed with C. olivaceum and incubated for H-Sd at 25°C (ie. until a good zone of inhibition was present). The zones of inhibition (approx. 1.5cm squares) were then aseptically removed from the agar plates and one of the following experiments were conducted in duplicate (controls of un-inoculated TSY agar was also done) : 1) An agar disc was placed in a sterile tube containing a sterile filter paper disc (so as to saturate the disc) and contents allowed to stand for 2Hb at room temperature. 2) Four agar discs were placed in a sterile tube containing 5ml of sterile distilled water. Tubes were then allowed to stand for 21h at room temperature, +/- vortexed for 1min, and contents then soaked onto a sterile filter paper disc. 3) An agar disc was placed into a glass petri dish containing a filter paper soaked with sterile distilled water on the bottom. A sterile filter paper disc was then placed on top of the agar disc and sterile air was carefully blown over the petri dish for better saturation of the filter disc. 57. A l l f i l t e r d iscs were then placed onto TSY agar plates, s p r a y e d with a spore suspension of Olive green mold, and incubated for 4-5d at 25°C (examined d a i l y for zones of i n h i b i t i o n ) . ii) Mesophilic conditions: Since the TSY agar plates showing i n h i b i t i o n against Ol i v e green mold (by Ba c i l l u s AOG) had been incubated at mesophilic temperatures (ie. 25°C), the i n h i b i t o r may only be produced by Ba c i l l u s AOG at this lower temperature range. Hence,Bacillus AOG was inoculated into 100ml of TSY broth and incubated for 2, 4, 6, and 8d at 25, 37, and 55°C i n a shaker waterbath. C u l t u r e s were then c e n t r i f u g e d at 8000rpm (10,400 x G; GSA rotor) i n a S o r v a l l RC2-B (0°C) for 35min and f i l t e r e d t h r o u g h a 0.22um millipore. The f i l t r a t e was then f r e e z e - d r i e d and reconstituted in eit h e r 1ml of cold s t e r i l e d i s t i l l e d water or 1ml of n-butanol. Samples (0.1ml) were soaked onto f i l t e r paper d i s c s , placed onto TSY agar plates and sprayed with C. olivaceum. Plates were examined d a i l y for zones of i n h i b i t i o n a f t e r 4d incubation at 25°C. 58. iii) Evaporation method: Cultures of Bacillus AOG were incubated in TSY broth for 7d at 25°C, 150rpm (New Brunswick Psychrotherm shaker). Following incubation, cultures were centrifuged at 8000rpm (10,400 x G) for 30min (GSA rotor) and then mixed 1:1 with n-butanol. Extraction (2 times) was carried out at room temperature by agititation in a large separatory funnel. The butanol layer was then removed and i concentrated (Brinkman Rotavapor R116) to approximately 10ml. This concentrate was centrifuged at 15,000rpm in a SS-34 rotor (27,000 x'G) for 20min and stored at 4°C for further analysis. 59. C. Temperature Stability 0.1ml of the n-butanol extracted antibiotic was placed into a series of 100 microlitre pipettes and given one of the following treatments in duplicate: 1) heated (dry) at 25, 50, 75, 100, 125, and 150°C for 1, 5, 10 and 15min (Despatch oven). or, 2) frozen at -15°C for 48h, and 3 months (Viking freezer). Following treatments, the pipette contents were placed onto filter paper discs on TSY agar and challenged for activity against C. olivaceum (25 °C ) . Heat treatments could not be tested at temperatures greater than 150°C (5min) because of evaporation/drying of the pipette contents at the higher temperatures. D. pH Stability The extracted inhibitor (in water) was adjusted to various pH levels (2-10) with either IN HC1 or 1N NaOH and tested for activity against C. olivaceum, using filter paper discs on TSY agar (25 °C ) . 60. E_. Solvent S o l u b i l i t y F r eeze-dried c u l t u r e s of B a c i l l u s AOG were reconstituted in cold s t e r i l e d i s t i l l e d water as outlined p r e v i o u s l y . One ml of a p a r t i c u l a r solvent (water, n-butanol, n-propanol, isopropanol, ethanol, methanol, acetone, p y r i d i n e , dioxane, e t h y l acetate, chloroform, toluene, hexane, benzene, petroleum ether, carbon t e t r a c h l o r i d e , or cyclohexanol) was then added to the 1ml of B a c i l l u s AOG e x t r a c t . The solvents were all of reagent, A.C.S. or U.S.P. grade. Samples were t h e n w o r t e x e d v i g o r o u s l y for 1min and then, c e n t r i f u g e d at 3000rpm in a SS-34 rotor (1085 x G; 0°C) for 15min. Following c e n t r i f u g a t i o n , the pH was determined ( F i s h e r pH paper) of the solvent e x t r a c t e d layer and 0.1ml (of solvent e x t r a c t e d layer) was placed onto a s t e r i l e f i l t e r paper d i s c . These di s c s were let stand in a s t e r i l e p e t r i d i s h for 12h at room temperature in o r d e r to evaporate excess solvent. 0.1ml of the solvent only was also added to a control d i s c . A l l experiments were conducted in d u p l i c a t e . Discs were then placed on a TSY agar plate, challenged with Olive green mold and incubated at 25°C for 4d. Following i n c u b a t i o n , zones of i n h i b i t i o n were measured and recorded. 61. F. Spectrum of Activity of Antibiotic Bacillus AOG was streaked once across a TSY agar plate and incubated for 20h at 55°C. Following incubation, various test organisms (Table 1) were streaked on the same TSY plate at right angles to the streak of Bacillus AOG. All test organisms had previously been grown on TSY agar for a minimum of 48h. Plates were incubated in duplicate at both 37 and 25°C for 4d. The antibiotic was concluded to be effective against those microorganisms which demonstrated a lack of growth in the vicinity of the Bacillus AOG streak after incubation. G_. Thin Layer Chromatography Purification of the antibiotic was initially conducted through thin layer chromatography. The solvents employed were all of reagent, A . C . S , or U.S.P. grade. Various solvent systems were investigated to determine the optimum separation/purification of the crude antibiotic mixture (Table 7). After extraction of freeze-dried extracts of Bacillus AOG with n-butanol as previously described, 3.0ml of extract was streaked across the entire origin of a 20 x 20cm Silica gel 60 preparative plate (2.0mm thickness) with no indicator (EM Reagents). These plates had been previously activated by heating for 48h at 100°C (Blue M, Illinois). The chromatography Table 1. Microorganisms tested for s u s c e p t i b i l i t y to an t i b i o t i c . Organism A T C C Gram negative Serratia marcescens 8100 Proteus vulgaris 13315 Escherischia coli 25922 Klebsiella pneumoniae 13883 Enterobacter cloacae 23355 Gram pos i t i v e Staphylococcus aureus 25923 Streptococcus pyogenes- 19615 Streptococcus lactis 19435 Streptococcus cremoris 19257 Bacillus subtilis 28281 Bacillus megaterium 25848 Yeasts Candida lipolytica 8862 Candida utilus 9256 Saccharomyces cerevisiae 7753 Saccharomyces cerevisiae 7754 Saccharomyces pastorianus 2 339 63. tanks were allowed to e q u i l i b r a t e with the developing solvent before each r u n . Development took place a t room temperature. The p r e p a r a t i v e chromatograms were developed with n-butanol - acetic acid (glacial) -water (60:20:20 V/V/V) for a distance of I5cm (approx. 12h). The plates were removed from the tanks, a i r d r i e d , and biol o g i c a l l y active bands (see below) ex t r a c t e d three times with n-butanol. 0.5ml of the biologically active bands were streaked across the o r i g i n of separate 20 x 20cm pla s t i c S i l i c a gel analytical plates (0.2mm thickness) with no indi c a t o r (Merck Kieselgel 60). The chromatograms were then r u n in a methanol - chloroform - 17% ammonium h y d r o x i d e solvent system (40:10:20 V/V/V) for a distance of 15cm (approx. 90 min).at-room temperature. R, values of appr o p r i a t e bands were calculated and recorded. Location and ch a r a c t e r i z a t i o n of T L C bands : i) a i r d r i e d chromatograms were examined under both short and long wave ul t r a v i o l e t l i g h t (ChromatoVue; U l t r a v i o l e t Products Inc., San G a b r i e l , C a l i f . ) . ii) b iologically active bands - to locate the biologically active bands, 2.5cm of a T L C plate was layered with a th i n section (approx. 0.3cm) of TSY agar, s p r a y e d with C. olivaceum and incubated at 25°C for 4d. Chromatogram sections were observed daily f or the presence of zones of i n h i b i t i o n . 64. iii) to check ii) - biologically active bands were also extracted from the chromatograms with n-butanol and 0.1ml was saturated onto filter paper discs (in duplicate). Discs were placed onto TSY agar, sprayed with Olive green mold, and incubated for 4d at 25°C. Plates were examined daily for the presence of zones of inhibition. iv) spray reagents - a) Ninhydrin: 0.15g of 1, 2, 3, indantrione monohydrate (Ninhydrin) (MCB Chemicals) was mixed with 50ml of n-butanol, followed by the addition of 1.5ml of glacial acetic acid (according to the method of Stahl, 1969). This reagent was sprayed across the TLC plate and then the plates were heated at 110°C until optimum color development occurred (approx. Imin). A yellow color indicated the possible presence of proline and hydroxyproline; whereas violet was representative of all other alpha amino acids. b) Rhodamine 6C: TLC plates were sprayed with a Rhodamine 6C (Img Rhodamine 6C dissolved in 100ml acetone) solution and observed under longwave ultraviolet light. This spray reagent indicates the possible presence of lipids (Stahl, 1969). c) Phenol-sulphuric acid: Chrom-atograms were sprayed with phenol-sulphuric acid reagent to detect the presence of sugars. This reagent was prepared by adding 3g phenol and 5ml concentrated r^SO^ in 95ml ethanol. Chromatograms were then sprayed, and heated for I5min at 100°C (Stahl, 1969). 65. d) Alpha-cy clodextr'm: Chrom-atograms were s p r a y e d with aZpha-cycIodextrin (30% ethanolic solution of aZpha-cyclodextrin), a i r - d r i e d , and placed in a closed tank containing iodine vapor. T h i s spray reagent indicates the presence of s t r a i g h t chain l i p i d s ( S t a h l , 1 969). H_. U l t r a v i o l e t Spectrum P u r i f i e d samples of Bands I (3.1mg) and II (5.0mg) (TLC ) in n-butanol were scanned under u l t r a v i o l e t l i g h t ( V a r i a n C a r y 210 Spectrophotometer) and data was recorded. The a b s o r p t i v i t y of the samples were also ca l c u l a t e d , u s i n g the equation: A = a b • c where "A absorbance a a b s o r p t i v i t y b li g h t path (cm) c concentration (mg/ml) 66. ]_. Column Chromatography i) lon-exchange chromatography: a) column pre p a r a t i o n : A cation exchange r e s i n - AG 50W X-8 with 100-200 mesh size (Biorad) was hydrated with d i s t i l l e d deionized water and poured into a 2.0 x 25cm glass column. T h i s r e s i n was converted to the H + form by washing with 150ml of IN HCI and t e s t i n g for low pH (0.5). The column was then r i n s e d with 200ml of deionized water for a fi n a l pH of 6.5. b) sample el u t i o n : 10ml of the sample ( B a c i l l u s antibiotic) in n-butanol was c a r e f u l l y layered onto the column. Elution was performed with a discontinous g r a d i e n t of 100ml of 0.01N, 0.02N, 0.05N, 0.08N, and 0.1N NH^OH. A column flow rate of 1ml/min was maintained at room temperature. Samples were collected on an Isco F r a c t i o n C o l l e c t o r . c) detection of sample: The O.D. of all tubes were read at U.V. 275um and 270um and data recorded ( V a r i a n C a r y 210 Spectrophotometer). 67. ii) Sephadex LH 20: a) column preparation: Sephadex LH 20 (Pharmacia Fine Chemicals), with a particle size of 25-100u was swollen overnight in boiled distilled water. Excess water was removed until a thick slurry resulted and this was then deaerated under vacuum. Chromatography was performed in a 2.5 x 30cm glass column. b) sample elution: 5ml of sample (Bacillus anti-biotic) in distilled water was carefully layered onto the column. The sample was eluted with 600ml of distilled water using a flow rate of 1ml/min. at room temperature. c) sample detection: Each tube was scanned under U.V. light (Varian Cary 210 Spectrophotometer) and its spectrum recorded. Fractions demonstrating a U.V. peak ( and similar U.V. scan) were pooled and freeze-dried. These extracts were then reconstituted with 1ml of n-butanol, saturated onto filter paper discs (0.1ml) and placed onto TSY agar plates. These plates were sprayed with C. olivaceum, incubated at 25°C and examined daily for the presence of zones of inhibition. 0.3ml of reconstituted sample was also streaked across the entire origin of a 7 x 20cm plastic Silica Gel 60 plate (0.2mm, no indicator). Chromatograms were then run in a butanol - acetic acid - water solvent system (60:20:20). Location of biologically active bands was performed as previously mentioned (Section G of Methods & Materials). 68. J_. Fluorescent Spectrum Biol o g i c a l l y active bands (from T L C analysis) were scanned on an Aminco-Bowman Spectrophotofluorometer and spec t r a recorded. K. Amino A c i d A n a l y s i s Amino acid analysis was performed on p u r i f i e d samples of the a n t i b i o t i c . Following e x t r a c t i o n from T L C plates as p r e v i o u s l y d e s c r i b e d , appropriate bands ( i e . biologically active) were d r i e d under a nitrogen f l u s h . These c r y s t a l s (lower T L C band, 2.1mg; upper T L C band, 1. 3mg) were then h y d r o l y z e d with p-toluenesulfonic acid in the presence of 3-(-2 amino-e t h y l indole) for 24h at 110°C according to the method of L i u & Chang (1971). Because of v e r y low sample amounts no SH b l o c k i n g of cys t e i n e was conducted, and only 1ml of IN NaOH was added to samples after h y d r o l y s i s . The digested samples were then f i l t e r e d t hrough an u l t r a f i n e s i n t e r e d glass f i l t e r . The f i l t e r had p r e v i o u s l y been re v e r s e d flushed with IN NaOH, d i s t i l l e d water, and neu t r a l i z e d with IN HC1; a fina l r i n s e was conducted with d i s t i l l e d water and then a i r d r i e d using acetone. 0.5ml of the f i l t e r e d samples were analyzed on a Phoenix Model 6880 (Phoenix Instruments, Phil.) amino acid analyzer u t i l i z i n g a singl e column elution system (Durram Chemical Corp., Palo A l t o , C a l i f . ) . ' 69. RESULTS AND DISCUSSION l_. Isolation of Thermophiles Antagonistic Towards Chaetomium olivaceum Initially, it was decided to isolate thermophilic microorganisms which would both inhibit C. olivaceum and support Agaricus bisporus using a medium which resembles actual compost substrates (ie. "synthetic compost medium"). The lignin content of the synthetic compost medium (ie. ball-milled wheat straw) was shown to possess 22.2% lignin. The control compost sample (ie. fermented) on the other hand, demonstrated a 30.1% lignin content. These data seemed to support the fact that mushroom composting results in an increase in the amount of lignin (Waksman S Nissen, 1931). The U.V. spectra of methanol extracted lignin from wheat straw and compost are shown in Figure 4. The peak in the U.V. region 275um corresponds to the phenolic content of the lignin (Aulin-Erdtman, 1949; Brauns, 1952). Although these data looked promising for further lignin research, as a selection protocol it (synthetic compost) was found to be an extremely lengthy and involved procedure. The greatest time employed was found to be in preparing the media - for example, ball-milling the wheat straw, having to add the nutrients extremely carefully for even moisture dis-tribution, and lignin analysis; also waiting for the A. bisporus mycelium to develop (2-3 weeks) on this medium (possibly due to lower amounts of F igure t. Ultraviolet spectra of l ign in extracted from wheat straw and compost. 71. lignin). Although this method was repeated twice, no C. olivaceum inhibition resulted with over forty thermophiles tested in this way. These factors forced a reconsideration of the medium and method to a revised selection protocol which resulted in a much more efficient approach to this problem. The second method involved spraying a spore suspension of Olive green mold directly onto all TSY plates following the initial dilution and incubation of compost samples (see Methods and Materials). This demonstrated an immediate reaction (ie. approx. 3d) between the C. olivaceum and any of the thermophiles on the TSY plates. The organisms capability to support Agaricus bisporus could be determined at a later time. After approximately four months of compost examinations using the revised selection procedure, ten thermophiles were isolated which showed varying degrees of antagonism towards Olive green mold on TSY agar plates (Figure 5). Three of the ten thermophiles (referred to as #1-3) were initially isolated during the use of method 1. 72. C d. Figure 5. Thermophiles (a - #9; b - #10; c - #4; d - #6) showing varying degrees of antagonism towards C. olivaceum on TSY agar. 73. M_. Identification of Isolated Thermophiles The next stage in this investigation was to determine the identity of the organisms which were causing inhibition of Chaetomium olivaceum {as shown on TSY agar). Pure cultures of the thermophiles were first subjected to classification according to their Cram reaction and cellular morphology. Microscopic observations demonstrated the organisms to range from Gram positive to Gram variable (Table 2), rod-shaped (approx. 1 x 4 ) (Figure 6), occurring generally in singles, pairs, and short chains. Endospore formation was also evident (subterminal to terminal), although the sporangia were not appreciably swollen by the spores in any of the cultures. Macroscopic observations showed the absence of mycelium oh any of the solid media tested. These observations implied that the ten isolated microorganisms belonged to the family Bacillaceae (Buchanan & Gibbons, 1975). Furthermore, since the thermophiles were shown to be facultative anaerobes (growth in sloppy agar along top and entire stab; growth in anaerobic jars) as well as being catalase positive (Table 3), it was concluded that they belong to the genus Bacillus. Members of the genus Bacillus can be defined (Wolf & Barker, 1968) as "rod-shaped organisms which are spore-bearing, usually Gram positive, catalase producing and capable of sporulating aerobically" (distinguishes Bacillus from some aero-tolerant Clostridia". The maximum growth temperature of these organisms was observed to Table 2. Microscopic observations and colony morphology of isolated thermophiles. Thermophiles #1-9 Thermophile #10 Gram stain Size - length (um) width (um) spore arrangement Appearance of colonies T S Y agar size shape margin pigmentation colony under reflected light transmitted light elevation other PDA agar pigmentation colony under reflected light transmitted light elevation other Agar slant growth rods (singles,pairs short chains) 2.0 - 4.8 .78 - 1.0 subterminal to terminal large (3-10mm) generally round irregular green/brown dull translucent relatively flat dull translucent flat no mycelium effuse rods (singles, pairs short chains) 1.7 - 5.31 .78 - .94 subterminal to terminal large (2-12mm) round to irregular smooth to irregular (undulate) tan to cream (slight green) shiny opaque umbonate/hilly extremely mucoid; some volcano-1 ike structures relatively dull opaque umbonate no mycelium effuse thermophiles #7, 9 were observed to be Gram negative. F i g u r e 6. Gram reaction of Thermophile # 10. Table 3. Properties of isolated thermophiles. Property Thermophiles #1-9 Thermophile #10 Motility Anaerobic growth Temperature Maximum Minimum Catalase Voges-Proskauer Methyl-red Resistance to lysozyme Growth in 10% NaCl : Growth at pH 5.7 Growth in 0.02% azide Acid from glucose arabinose xylose mannose Hydrolysis of starch Utilization of urea citrate propionate N 0 3 " to N 0 2 " Production of dihydroxyacetone indole Deamination of phenylalanine Decomposition of casein tyrosine Litmus milk 60 °C 15°C 65°C 15°C alkaline clot reduction alkaline clot reduction Mannitol salt 77. be 60 to 65°C. According to Buchanan £ Gibbons (1975) and Gordon et al (1973), there are only five species of the Bacillus genus which are known to be capable of growth over 50°C (Table 4). B. stearothermophilus has a maximum growth range of 65 - 75°C; B. coagulans, 55 - 60 (65) ° C ; B. brevis, 40 - 60°C; B. licheniformis, 50 - 55°C; and B. subtilus 45 -55°C. Thermophiles #1 through #9 were observed to be the same (or extremely closely related) species of organism - as shown by biochemical and cellular characteristics (Table 2, 3). There was only a difference in Gram reaction with cultures #7 and #9 as compared to the others in this series; this discrepancy may be due to differences in age and/or staining technique. Moreover, thermophiles #1 through #9 all differed from #10 in such properties as maximum temperature of growth, sugar fermentation pattern, utilization of citrate, nitrate reduction, colony morphology and were thus considered distinct species. It was concluded from the above facts (ie. cellular and biochemical properties) that all of the isolated thermophiles belonged to the species Bacillus coagulans (or a closely related variant of B. coagulans). This decision is based on the following: 1. B. coagulans is the only known thermophile which is capable of growth in 0.02% sodium azide. 2. Ability of isolated thermophiles to grow in acid media. Table 4. Summary of properties of known thermophilic Bacillus species. Property Thermophile B. licheniformis B. coaqulans B. brevis B. stearothermophilus B. subtilus size - width (u) 0.6 to 0.8 0.6 to 1.0 0.6 to 0.9 0.6 to 1.0 0.7 to 0.8 - length (u) 1.5 to 3.0 2.5 to 5.0 1.5 to 4.0 2.0 to 3.5 2.0 to 3.0 Gram stain Gram + Gram +/v Gram +/v Gram +/v/- Gram + spore formation central/ subterminal or central. / subterminal / central / paracentral; terminal ; subterminal / terminal; paracentral; slight swelling slight to no terminal; definite swelling of slight to no of sporangia swelling of swelling of sporangia swelling sporangia sporangia of sporangia motility + + + + + catalase + + + V + anaerobic + + - - -temperature maximum 50-55 55-60(65) 40-60 65-75 45-55 °C) - minimum 15 15-25 10-35 30-45 5-20 V.P. + + - - + resistance to lysozyme - V - V NaCl 10% + - - - + pH 5.7 + + V - + Table 4. - Continued Property Thermophile acid from - glucose arabinose xylose mannose starch hydrolysis utilization of citrate utilization of propionate NO. NO. '3 ""2 dihydroxyacetone indole phenylalanine: deamination decomposition of - tyrosine casein B. lichenformis B. coagulans B. brevis B. stearothermophilus B. subtilus ND ND ND + v v V V V ND V + V V V ND V ND ND ND ND - not done + - positive for 90-100% of strains - negative for 90-100% of strains v - character inconstant 80. 3. B. lichenif'ormis and B. coagulans are the only thermophiles which are capable of growth under anaerobic conditions. 4. Microscopic and biochemical properties of the isolated thermophiles closely resembles those of B. coagulans; exceptions to this are the isolates' ability to grow in high salt concentrations (10% NaCl) and their resistance to lysozyme. These dissimilarities may be due to biochemical variations within the species which are known to occur (Campbell & Sniff, 1959; Humphreys & Costi-low, 1957; Marshall S Beers, 1967), variation due to different basal media used during testing (Gordon et al, 1973), plus the observations of Allen (1953) who noted that the characteristics of the thermophilic Bacillus species tended to change on continued culture in the laboratory. B. coagulans has been reported to exist in a variety of morphological types (Wolf& Barker, 1968). Bacillus coagulans was first described in the literature by Hammer in 1915, and has since been classified into two distinct morphological types. Smith et al (1952) defined group I as sporangia not appreciably swollen by oval spores. Hence, the organisms of this study were classi-fied as belonging to group I. The most fundamental characteristics of B. coagulans are its acido-philic and thermophilic properties (Wolf S Barker, 1968). Virtually all types grow at 60°C and are capable of initiating growth at pH 5.3. Growth at 81. low temperatures is affected both by the medium and nature of the inoculum, spores prov ing more responsive to lower temperature than vegetative cel ls . Hence it is the maximum temperature which may be of greatest s ignif icance in classif ication of Bacil lus thermophiles (Wolf & Sharp , 1981). Essent ia l ly, B. coagulans is a facultat ive;thermophile, growing well at 45 - 55°C. Dur ing the period of identif icat ion, it was found that thermophil ic Bacil lus species #1 through #9 demonstrated a much lower antagonism towards C. olivaceum on T S Y agar. Th i s var ied from total lack of inhibition of Ol ive green mold to extremely minimal antibiotic capabi l i ty. It seemed that these cultures had or were undergoing a possible mutation process du r ing the cult ivation on laboratory medium. Fresh isolates of thermophiles #1 through #9 which had been stored at 3°C on T S Y agar slants were regrown at 55°C on fresh medium. These cul tures were also soon shown to have lost their antagonism against Ol ive green mold (usually after a few transfers on T S Y plates) . However, thermophil ic Bacil lus #10 maintained an excellent inhib i tory effect against C. olivaceum (F igure 7) since its initial isolation and also du r ing the identif ication protocol. T h u s , only Bacillus coagulans #10 was used for any fur ther experimentation (hereinafter re ferred to as Bacil lus AOG - "An t i -O l i ve g r e e n " ) . F igure 7. Inhibition of Ol ive green mold by Bacil lus A O G . 83. III. , Cultivation of Agaricus bisporus with the Thermophile- Bacillus AOG To determine if Bacillus AOG would support the mushroom, Agaricus bisporus, as well as protect it from damage by Olive green mold, this thermophile was inoculated into two kinds of culture media. One medium was conventional - consisting of standard phase II mushroom compost; and the other was hydroponic - consisting of liquid substrates absorbed onto an inert physical support, vermiculite. 1. Conventional methods: Figure 8 represents the rate of mushroom mycelial development in standard compost over a seven day period. As can be seen, when the Bacillus was added to the compost, the rate of mycelial development in the trays was enhanced. In addition, the Bacillus AOG exhibited a definite inhibitory effect on the development of Olive green mold (F test on slopes p<.01). This biological protection was further indicated by a significant 86.5% increase in mushroom yield of the trays containing Bacillus AOG and Olive green mold over that of the trays containing Olive green mold only (Figure 9) (analysis of variance p<.05). Futhermore, trays with only C. olivaceum produced fruitbodies a full week later than those with Bacillus AOG and C. olivaceum 84. 2.5 4-• CONTROL • BACILLUS ADDED OCHAETOMIUM ADDED • CHAETOMIUM AND BACILLUS ADDED 2.0 4-1.5 4-1.0 4-0.5 4-0.0 DAYS Figure 8. Mycelial development in standard mushroom compost. 85. 175 •+- 1 1 CONTROL BACILLUS ADDED BACILLUS AND CHAETOMIUM ADDED CHAETOMIUM ONLY Figure 9. Yield of mushrooms in compost. 86. together. The observation that initation and development of fruitbodies were not retarded shows that Bacillus AOG had no apparent inhibitory effect on organisms such as P. putida, known to stimulate the formation of sporophores (Nair & Fahy, 1972). As demonstrated in these experiments, the application of Bacillus AOG may eventually form an effective biological control method (against Olive green mold) in the commercial production of the mushroom. 2. Hydroponic methods: The hydroponic series of experiments utilized either 2% (w/v) Malt extract or a liquid compost solution (containing 47% total solids) absorbed onto a carrier material of sterile vermiculite. Vermiculite (hydrated magnesium aluminum-iron silicate) is a common sorbent used in many hydroponic systems (Douglas, 1976; Resh, 1978) because of its excellent qualities. It is lightweight, neutral in reaction with good buffering properties, and is capable of absorbing large quantities of water or nutrients. Also, it has a relatively high cation exchange capacity and thus can hold nutrients in reserve and later release them. Vermiculite contains some magnesium and potassium (essential nutrients for mycelium production and sporophore formation) which are available to mushrooms. These factors seem to make vermiculite 87. a good choice for hydroponic c u l t i v a t i o n of A. bisporus. As can be seen from F i g u r e 10, when the B a c i l l u s was added to hydroponic t r a y s i n 2% Malt e x t r a c t , a remarkable improvement i n the rate of mycelial development o c c u r r e d (p<.01). The improved rate of mycelial growth is shown more v i v i d l y i n the resp e c t i v e photographs of the hydroponic t r a y s . F i g u r e 11 represents a t r a y with only Chaetomium olivaceum added - as can be seen, r e l a t i v e l y poor spawn growth is evident; F i g u r e 12 represents the effect of the B a c i l l u s AOG and Olive green mold present at the same time. As these r e s u l t s show, the mush-room mycelia f l o u r i s h e d i n the presence of the B a c i l l u s . Mushroom y i e l d s from the 2% Malt e x t r a c t experiments ( F i g u r e 13) conta i n i n g O l i v e green mold o n l y , showed complete f a i l u r e of any A. bisporus f r u i t b o d y formation. Furthermore, al l t r a y s without B a c i l l u s AOG (ie. unfermented 2% Malt extract) showed v i s i b l e s i g n s of contamination by an uni d e n t i f i e d blue mold. However, the crop y i e l d s from t r a y s containing the B a c i l l u s AOG were maximum - even in the presence of the Chaetomium mold. The r e s u l t s for hydroponic c u l t u r e with l i q u i d compost demonstrated s i g n i f i c a n t biological control d u r i n g both the mycelial growth phase (p<.01) ( F i g u r e 14) and d u r i n g f r u i t i n g of the mushroom as the yields demonstrate ( F i g u r e 15) (analysis of variance p<.05). T r a y s with only the competitor C. olivaceum added d i d not produce mushrooms. However, similar to the 2% Malt experiments, the maximum y i e l d o c c u r r e d for t r a y s containing the B a c i l l u s AOG and C. olivaceum together. Furthermore, 88. 2.5 + • CONTROL • BACILLUS ADDED OCHAETOMIUM ADDED D BACILLUS AND CHAETOMIUM ADDED 2.0 4-E u cc \-LLI 1.5 5 i-» LU U >-0.5 -f-0.0 -I DAYS Figure 10. Mycelial development in 2% malt extract. 89. Figure 11. Poor spawn growth in hydroponic tray with only Ol ive green mold present. .Figure 12. Improved mycelial development due to the biological protection of Bacillus AOG against C. olivaceum. 90. Figure 13. Yield of mushrooms in 2% malt. 91 2.5 -h 2.0. o NON-STERILE CONTROL • STERILE CONTROL • BACILLUS ADDED o CHAETOMIUM AND BACILLUS ADDED A CHAETOMIUM ADDED E u cn Ui \~ UJ < LLl U > 1.5-U 1.0-r-0.5 0.0 DAYS Figure 14. Mycelial development in liquid compost. 18.0 -h j | CONTROL [ffl BACILLUS ADDED H BACILLUS AND 15.0 4- CHAETOMIUM ADDED Bjggj CHAETOMIUM ONLY ^ 12.04 Q _1 UJ >-9.0 -f-6.0 3.0 4-N o n -s t e r i l e S t e r i l e Figure 15. Yield of mushrooms in liquid compost. 93. trays with Bacillus AOG in liquid compost were shown to produce the earliest occurring flushes as compared to any other medium. Overall yields in liquid compost were substantially larger than vermiculite trays containing 2% Malt extract. This is probably due to a much greater amount of essential nutrients (in the required form) initially present in the compost - as compared to those in 2% Malt extract (Fermor, 1982). This is substantiated by the carbohydrate analysis of 2% Malt extract during fermentation by Bacillus AOG. As shown in Figure 16, after the fourth day of incubation there is less than 0.05% of available carbohydrate left. Hence, one might conclude that A. bisporus obtained its carbon nutrition through utilization of the thermophile Bacillus AOG (or its products). It has been previously proven (Turner, 1977) that Agaricus can synthesize all the needed enzymes for use of microorganisms as a food source. These results clearly show that Bacillus AOG readily supports both mycelial growth and fruiting of the mushroom. Although liquid compost was the most successful hydroponic medium employed - the yields were still much lower than the conventional experiments. This could be explained by a lack of nutrients during the pinhead formation and/or subsequent fruitbody production stages in the hydroponic medium. In other words, mycelial development was shown to be as good in liquid compost as in the conventional trays (Figures 8 S 14) during the spawn run. Therefore, it seems the initial 500ml of liquid compost/tray (ie. water soluble nutrients of 21 18 72 HOURS OF FERMENTATION 96 Figure 16. Fermentation of 2% malt extract by Bacillus AOG. 95. compost) was largely depleted for mycelial development and thus the residual nutrients were insufficient for larger mushroom yields. How-ever, this could easily be corrected in future endeavours by replenishing the vermiculite beds prior to casing with fresh nutrients (or by improving the extraction protocol of the compost). These experiments clearly show the benefits resulting from selective protection (biological control) through controlled fermentation of the nutrient substrate. The successful use of hydroponics coupled with pure cultures of microorganisms are important factors in the micro-biological development of this fermentation process. Compost manufacture and materials are evaluated to cost 20 - 25% of the total mushroom production expenditures (Royse & Schisler, 1980). The annual consumption of horse manure in the U.S.A. now exceeds 350,000 tons and is quickly approaching the supply limit. This is reflected in a doubling of the horse manure price over the past two years (Hatch & Finger, 1979). These costs imply that the mushroom industry will require continued improvements in culture systems to stay competitive with other agricultural crops. However, the problem worsens by the ever changing composition of the manure (ie. manure from different stables) and seasonal variations in temperature and other environmental changes (Huhnke, 1970). These situations cause control of the already complicated composting process to be extremely difficult; and this leads to the production of varying grades of composts which create considerable fluctuations in the mushroom yield and often to total crop 96. failure. Furthermore, on many larger intensive units, the disposal of used compost frequently poses serious problems (eg. mushroom worker's lung disease; Kleyn et al, 1981). Out of necessity, less horse manure will be utilized in the future years - new forms of raw materials will have to be substituted. Therefore increasing attention is being given to systems of recycling/replenishing (eg. hydroponics) the nutrients which have been utilized by the mushroom so that they can be reused in the succeeding cycle. Dramatic improvements in composting will be imperative to contribute not only to standardization and control, but also to the economics of culture. With the use of hydroponic methods, the substrate no longer depends on the seasonal weather, and complete regulation of the substrate composition and its reproducible quality are virtually guaranteed. Cultures are healthier due to total elimination of all diseases and pests originally present in the substrate; moreover there have been several reports in North America of botulism poisoning due to consumption of canned mushrooms (Clostridium spores are worlds-wide in distribution'j-in soil "and compost).. The continous delivery of horse manure is no longer necessary and the grower is independent of its production. The bad smell and fumes of ammonia produced from the compost stacks are eradicated, thus facilitating the employment of workers. As neighbors are no longer disturbed by smell, mushroom enterprises can now be created near residential areas. In view of these advantages, it seems well worthwhile to adop the new procedure (thermophilic bacterial liquid feeding) as an .economic approach to the fermentation of Agaricus bisporus. 97. IV. A n a l y s i s of I n h i b i t o r Produced by Bac i l l u s AOG The next stage i n th i s thesis i n v e s t i g a t i o n was to determine the nature of the i n h i b i t o r produced by Bacil l u s AOG against Chaetomium olivaceum (as seen on TSY agar p l a t e s ) . A. Effect of pH : The f i r s t step i n analysis of the Baci l l u s AOG in h i b i t o r was to determine i f a s i g n i f i c a n t change i n pH was invo l v e d on TSY media (ie . since B. coagulans is a known ac i d o p h i l e ) . T h i s was done by measuring the pH of TSY agar plates at various locations afte r growth of B a c i l l u s AOG (and challenge with C. olivaceum); and by a separate pH growth s t u d y of Oliv e green mold. The pH of the TSY agar plates was shown to be r e l a t i v e l y constant throughout the en t i r e agar media (pH 8.8). Furthermore, C. olivaceum was observed to be capable of growth throughout an exte n s i v e pH range (4-10) and thus would not be i n h i b i t e d at pH 8.8. Good growth at both h i g h and low pH levels by Olive green mold has also been reported by Beach (1937). These data seem to c l e a r l y conclude that a change i n pH was not the responsible e ^ agent for i n h i b i t i o n of Chaetomium. 98. B_. Extraction of Antibiotic: Experiments were now designed to determine if the inhibitor /antibiotic^ (produced by Bacillus AOG) could be extracted from cell-free extracts of fermentation medium. i) Thermophilic extraction: Bacillus AOG was first grown at therm-ophilic temperatures (55°C) in TSY broth for periods of 2-5d. Following filter sterilization and concentration of the samples, attempts were made to extract the antibiotic compound using distilled water or 50% ethanol. However, when these extracts (on filter paper discs) were challenged for activity against C. olivaceum, no inhibition of Olive green mold occurred. Many Bacillus spp. are known to produce large quantities of antibiotic compounds at the sporulation stage of growth (Sadoff, 1972). Hence, a soybean meal fermentation media was utilized to attempt to stimulate extensive spore formation in Bacillus AOG. After 6-8d incubation at 55°C in soybean meal. Gram staining revealed the presence of large ovoid spores in Bacillus AOG. Extraction of the sterile culture filtrates was carried out with n-butanol. This method of extraction (ie. n-butanol) closely resembles According to Wakman's definition (Stahl, 1969), antibiotics are sub-stances which are formed by microorganisms and which kill off other microorganisms or inhibit their growth. This definition can thus be applied to the product produced by Bacillus AOG (against C. olivaceum). 99. the procedures commonly employed for isolation of many Ba c i l l u s sp. p r o d u c i n g a n t i b i o t i c s . However, similar to the above e x t r a c t i o n these c e l l - f r e e e x t r a c t s also showed no a c t i v i t y against Ol i v e green mold when tested on T S Y agar. From the poor r e s u l t s with broth media, i t was decided to t r y to e x t r a c t the i n h i b i t o r d i r e c t l y from the TSY agar plates. T h i s was done by removing asep t i c a l l y the i n h i b i t i o n zone produced by Bacil l u s AOG against C. olivaceum (on T S Y agar) and subsequently attempting to leach the an t i b i o t i c out of the agar onto f i l t e r paper d i s c s . Only the experimental t r i a l s u s i n g s t e r i l e a i r to concentrate the e x t r a c t onto the s t e r i l e f i l t e r d i s c s was reasonably s u c c e s s f u l (Table 5K . Other experiments were found to be eith e r contaminated (ie . due to Ba c i l l u s AOG spores p r e s e n t ) , or demonstrated no antagonism towards C. olivaceum ( p r o b a b l y due to low amounts of compound absorbed onto d i s c s ) . These methods were repeated twice with no better y i e l d i n g r e s u l t s . ii) Mesophilic e x t r a c t i o n : It was suggested that since plates showing i n h i b i t i o n of O l i v e green mold by Bac i l l u s AOG had been incubated at 25°C, the production of th i s a n t i b i o t i c compound may only be created d u r i n g mesophilic growth temperatures. Hence, B a c i l l u s AOG was grown in T SY bro t h at 25 and 37°C for periods r a n g i n g from 2-8d. Following Table 5. Extraction of inhibitor from TSY agar discs. Experiment Zone of inhibition (mm) 1) control - uninoculated TSY agar 0 2) agar disc + filter paper only 0 3) agar disc with 5 ml distilled water (+/- vortex) 0 4) saturation of disc utilizing sterile air 22 ^ v e . of 2 plates 101. harvesting, ceil-free concentrates were extracted with n-butanol and saturated into filter paper discs. As shown in Figure 17, the production of the antibiotic was indeed only produced during the lower growth temperatures by Bacillus AOG. It might be postulated that the organism Bacillus AOG only synthe-sizes this antibiotic at a mesophilic range of temperature because of certain thermophilic requirements at higher temperatures. In other words, the inhibitor may be a needed component of the cell wall under the thermophilic requirements (55°C) but is not required at mesophilic temp-eratures (25, 37°C) and thus becomes expelled from the cell. AH further extractions of the antibiotic were conducted with cultures-at 25°C in TSY broth for 7d incubation. Extraction and concentration of the antibiotic with n-butanol can also be done directly (ie. without freeze-drying) by evaporation/concentration of the fermentation broth on a Rotavapor apparatus (see Methods S Materials). Successful extraction by evaporation is especially suited for possible industrial applications (ie. excessive costs are employed for freeze-drying). Figure 17. Inhibition of C. olivaceum from cell-free extracts of Bacillus AOG. 103. C_. Temperature and pH Stability of Antibiotic: The extracted antibiotic was shown to be stable over a wide range of both pH and temperature. It was found to be active (towards C. olivaceum) over the entire range of pH (2-10) and temperature (-15°C to 150°C) tested. Even after storage for 4 months at -15°C no loss in activity was observed. Hence, the antibiotic appears to be capable of growth over an extensive spectrum of physical conditions. D. Solvent Solubility: Studies on the solvent solubility of the antibiotic demonstrated it to be soluble in polar solvents (Table 6); water alone, n-butanol, n-propanol, isopropanol, ethanol, methanol, dioxane, pyridine, and acetone; whereas it is insoluble in chloroform, ethyl acetate, toluene, hexane, benzene, petroleum ether, and carbon tetrachloride. This antibiotic thus seems to be hydrophilic in nature. 104. Table 6. Solvent s o l u b i l i t y of a n t i b i o t i c . Solvent 1 pH Zone of Inh i b i t i o n ( A v g of duplicate plates) (cm) water 7.0 3.3 acetone 7.5 3.4 n-propanol 7.5 3. 3 p y r i d i n e 7.6 3.0 n-butanol 7.0 3.0 isopropanol 7.5 3.0 ethanol (100%) 7.5 3.0 methanol 8.0 2.9 dioxane 7.5 2.9 e t h y l acetate 6.5 0.0 chloroform 6.5 0.0 toluene 6.5 0.0 hexane 6.5 0.0 benzene 7.0 0.0 petroleum ether 6.5 0.0 carbon t e t r a c h l o r i d e 6.5 0.0 cyclohexanol 6.5 4.01 ^control plates ( i e . same extent. cyclohexanol only) also i n h i b i t e d C. olivaceum to the 105. E_. Spectrum of A c t i v i t y : It was found that the an t i b i o t i c demon-str a t e d no sup p r e s s i o n of growth of any Gram negative organisms or of the Gram po s i t i v e cocci tested. However, when tested a g a i n s t 7 Bacillus subtilus and B. megaterium it was found to be quite potent ( F i g u r e 18). Furthermore, the growth of the fungus Candida lipolytica. was also i n h i b i t e d . T h erefore thi s a n t i b i o t i c can be used e f f e c t i v e l y not only against Chaetomium olivaceum but other microorganisms as well. F. T h i n L a y e r Chromatography: Because of it s h i g h r e s o l v i n g power and speed, t h i n layer chromatography lends i t s e l f well to the separation and i d e n t i f i c a t i o n of ant i b i o t i c s ( S t a h l , 1969). T h e r e f o r e , p u r i f i c a t i o n of the a n t i b i o t i c produced by Ba c i l l u s AOG was i n i t i a l l y c a r r i e d out by T L C on S i l i c a Gel plates. Table 7 shows the r e s u l t s of various solvent systems used to determine the optimum separation of the c r u d e an t i b i o t i c e x t r a c t . The presence of p y r i d i n e i n any solvent system was shown to destroy the biological a c t i v i t y of the a n t i b i o t i c and hence not f u r t h e r used. It was found that the best resu l t s o c c u r r e d with an n-butanol - acetic acid (glacial) - water (60:20:20) solvent system followed by p u r i f i c a t i o n in a methanol - chloroform - 17% ammonium h y d r o x i d e (40:40:20) T L C system. These solvent mixtures are two of the most widely u t i l i z e d systems i n separation of ant i b i o t i c s ( S t a h l , 1969). 106. a) B. subtilus b) C. lipolytica c) S. aureus cj) B. megaterium e) S. lactis f) C . lipolytica Figure 18. Spectrum of a c t i v i t y of Bacillus AOG against various microorganisms. Table 7. Solvent systems investigated to determine optimum TLC separation of crude antibiotic. Solvent System Observations n-butanol:glacial acetic acid:water (60:20:20) methanol:chloroform: 17% ammonium hydroxide (40:20:20) n-butanol: n-propanol: water (60:20:20) n-butanol :n-propanol: water :glacial acetic acid (60:20:10:5) n-propanol:water (70:30) methyl ethyl ketone:pyridine:water: glacial acetic acid (70:15:15:2) n-butanol:pyridine:glacial acetic acid:water (30:20:6:24) n-propanol:water:pyridine (70:15:15) ethyl acetate:pyridine:glacial acetic acid:water (50:10:10:10) chloroform:methanol (50:50) optimum separation fair to good separation pyridine found to cause loss in biological activity no movement of sample from origin methanol:chloroform (3:97) ethyl acetate:methanol (87:13) 108. As p r e v i o u s l y mentioned (Methods £ M a t e r i a l s ) , f r e e z e - d r i e d e x t r a c t s of Ba c i l l u s AOG (i n n-butanol) were streaked across a S i l i c a Gel 60 chromatogram and r u n i n a butanol - acetic acid - water T L C system. To locate the biologically active bands, a portion of the a i r d r i e d plate was layered with T S Y agar, sprayed with Ol i v e green mold and incubated at 25°C. Two Ul t r a v i o l e t l i g h t p o s i t i v e ( v i s i b l e under both short and long wave - however, b r i g h t e r under short) bands with values of .46 and .52 (analytical 0.2mm plate) res u l t e d in i n h i b i t i o n of C. olivaceum. T h i s i n h i b i t i o n was shown to be extremely stable on the T L C plates. The lower band (Band I) appeared as a broad yellow band under short wave U.V. l i g h t ; whereas the upper band (Band II) appeared as a narrow l i g h t blue band. Furthermore, Band I was observed to produce bubbles when it was layered with T SY agar - this may poss i b l y indicate gas production due to the possible presence of perhaps a ca r b o x y l group or C 0 2 group i n the lower T L C band. Bands I and II when sprayed with n i n h y d r i n reagent produced a : violet color reaction i n d i c a t i n g the possible presence of amino acids ( S t a h l , 1969). Presence of l i p i d s was not observed in either band when T L C chromatograms were sprayed with either Rhodamine 6G or alpha-c y c l o d e x t r i n . A phe n o l - s u l p h u r i c acid s p r a y resu l t e d in B a n d T appearing li g h t brown and Band II a medium brown color afte r heating plates for 15min at 100°C. These color reactions may denote the presence of sugars i n the two biologically active bands ( S t a h l , 1969). When p r e p a r a t i v e plates (2.0mm thickness) were used for p u r i f i c a t i o n 109. in butanol - acetic acid - water, chromatograms were air dried after the first run, and developed a second time in this same solvent (in same direction) which resulted" in better separation of the bands. Revalues of .66 for Band I and .73 for Band II resulted. The two biologically active bands (from preparative plates) were then scraped off the chromatogram,.extracted in n-butanol and layered across separate analytical Silica Gel plates (0.2mm thickness) for further purification in methanol - chloroform - 17% ammonium hydr-oxide. Earlier observations showed poor separation had occurred in the methanol - chloroform - 17% ammonium hydroxide system when using a preparative TLC plate. R^  values in methanol - chloroform - 17% ammonium hydroxide were: for Band I, .27; and for Band II, .32. Bands on these plates were also shown to be biologically active and ninhydrin positive when layered with TSY agar and challenged with Olive green mold; or sprayed with ninhydrin reagent respectively. Purification of the Bacillus AOG antibiotic by thin layer chroma-tography was shown to be an extremely long and tedious process (and expensive). This is especially true when analytical TLC plates of only 0.2mm were used for the final purification stage. This resulted in extremely-low amounts of pure inhibitor being isolated. For example, approximately 20 analytical Silica plates would result in only perhaps a yield of 1-2mg of antibiotic crystals. Hence, it was decided to utilize column chromatography techniques in an attempt to isolate larger quantities of the antibiotic. 110. C. Column Chromatography: i) Ion-exchange: Because of the possible presence of amino acids in the a n t i b i o t i c (as indicated by n i n h y d r i n reaction on T L C plates) ion-exchange chromatography was employed as the f i r s t column method of separation. The a n t i b i o t i c e x t r a c t (in n-butanol) was c a r e f u l l y layered onto a cation exchange r e s i n ( H + form) with a high cross l i n k i n g (X-8) capacity. T h i s high cross l i n k i n g made it especially suited for separation of amino acid and small peptide compounds (according to manufacturer's s u g g e s t i o n ) . However, as shown by the elution p r o f i l e ( F i g u r e 19) of the a n t i b i o t i c e x t r a c t , the sample d i d not b i n d to the column and was washed out d u r i n g the f i r s t elution with d i s t i l l e d water. Non-binding may have been due to the sample having no ionic charge, or possessing the same charge (-) as the functional group of the res i n ( i e . S 0 3 ). In other words, d u r i n g ion-exchange chromatography, neutral molecules and those having the same charge as the functional group (ie. of the column) flow through the column and are separated from any sorbed ions (ie. those with a + charge in t h i s case). Or, perhaps non-binding may have been due to the sample being a much l a r g e r compound than i n i t i a l l y b elieved. ii) Sephadex LH 20: F u r t h e r column chroma-tography of the a n t i b i o t i c was conducted with a Sephadex LH 20 type 0 U 8 12 16 20 21 28 32 125 FRACTION NUMBER Figure 19. Antibiotic elution profile in cation exchange resin. 112. r e s i n . Sephadex LH 20 can be used i n polar s o l v e n t s , and is commonly employed for separation of biologically active substances, natural and s y n t h e t i c polymers, and low molecular weight solutes ( J o u s t r a et a l , 1967). As well as separating compounds according to size LH 20 poss^ esses both h y d r o p h i l i c and l i p o p h i l i c p r o p e r t i e s , and can be used for p a r t i t i o n chromatography. The B a c i l l u s AOG e x t r a c t (in d i s t i l l e d water) was layered onto the Sephadex LH 20 column and eluted with d i s t i l l e d water. The use of d i s t i l l e d water as an eluant f a c i l i t a t e d the f r a c t i o n s c a p a b i l i t y of later being f r e e z e - d r i e d for concentration. A l l tubes were scanned t h r o u g h -out the U.V. spectrum and the f r a c t i o n s from 5 peaks (demonstrating similar U.V. spectra) w,ere pooled and f r e e z e - d r i e d . T h i n l a y e r chromatography of the r e c o n s t i t u t e d f r a c t i o n s revealed biological a c t i v i t y (against C. olivaceum) from only one set of pooled fra c t i o n s (tubes 16-20). These two biologically a c t i v e bands were v i s i b l e under U.V. l i g h t and were also n i n h y d r i n p o s i t i v e (ie. similar to previous T L C r e s u l t s ) . However, it seems Sephadex LH 20 d i d not separate the two bands (Band I and II) to any greater extent than u t i l i z i n g a s i n g l e p u r i f i c a t i o n stage with T L C in butanol - acetic a c i d -water ( i e . similar R^ . v a l u e s ) . Since optimum p u r i f i c a t i o n was necessary for f u r t h e r analysis of the a n t i b i o t i c , it was therefore decided to continue with t h i n layer chromatography as the method of p u r i f i c a t i o n . One should keep in mind however, that Sephadex LH 20 by no means was proven i n e f f e c t i v e as a chromatography technique for the B a c i l l u s AOG a n t i b i o t i c . T h i s r e s i n could potentially be used in f u t u r e endeavours 113. at large scale p u r i f i c a t i o n of the ant i b i o t i c with perhaps c e r t a i n modif-ications. For example, experimental t r i a l s with v a r i a t i o n s i n sample s i z e , column s i z e , and type of eluant used might re s u l t in better separation of the bands as compared to that witnessed in thi s i n i t i a l s t u d y . H_. U l t r a v i o l e t Spectrum A n a l y s i s : F i g u r e 20 shows the u l t r a v i o l e t s pectra of p u r i f i e d Bands I and II . Band I demonstrated a f a i r l y broad peak at 275mu and a smaller peak at approximately 288mu. Band II was observed to consist of a singl e peak at 270mu. The a b s o r p t i v i t y coefficients were .835 and .401 mgmlcm 1 for Bands I and II r e s p e c t i v e l y . I_. Fluorescent Spectrum A n a l y s i s : F i g u r e 21 represents the fluorescent spectrum of T L C Band I. As can be seen, a single peak o c c u r r e d at 360um -with a relative i n t e n s i t y of 42. There was no fluorescent spectrum detectable for Band II. T h i s is in agreement with the re s u l t s of amino acid analysis (Section J) in which t r y p t o p h a n was not found in Band II (ie. t r y p t o p h a n is the major amino acid responsible for fluorescence). Figure 21. Fluorescent spectrum of T L C band I. 116. J . Amino A c i d A n a l y s i s : Amino acid analysis of Bands I and II from T L C chromatography revealed the i n h i b i t o r produced by Ba c i l l u s AOG to be a peptide a n t i b i o t i c (Table 8). Both bands were r e l a t i v e l y similar in amino acid composition. They both contained h i g h amounts of t y r o s i n e , s e r i n e , a s p a r t i c and glutamic a c i d s . Band I however, contained approximately three times the amount of phenylalanine as compared to Band II. Furthermore, Band I contained small amounts of l y s i n e , h i s -t i d i n e , t r y p t o p h a n and pro l i n e - these amino acids were not detected in Band II. Alt h o u g h cysteine was not SH blocked p r i o r to h y d r o l y s i s (due to small amounts of compound) not even a small peak was apparent for the presence of thi s amino a c i d . Furthermore, the breakdown products of cys t e i n e ( c y s t e i c acid) from h y d r o l y s i s with p-toluenesulfonic acid were shown not to be present. Unusual amino acids or peaks which could not be accounted for were also not found. T h i s amino acid p r o f i l e also indicates the an t i b i o t i c to be r e l a t i v e l y acidic in nature. T h i s feature might be responsible for the e a r l i e r observations (Section C) associated with ion-exchange chromatography (ie. non-binding to r e s i n ) . These r e s u l t s pose many i n t e r e s t i n g questions r e g a r d i n g the peptide an t i b i o t i c s produced by Baci l l u s AOG. For example, why does the organism produce two d i s t i n c t a n t i b i o t i c s t r u c t u r e s , why are they synthe-sized (especially at only mesophilic temperatures), and are these Table 8 . Amino acid analysis of Bands I and II. Band I (g/IOOg amino acid) Aspartic acid 19.90 Phenylalanine 18.90 Tyrosine 14.51 Serine 14.19 Glutamic acid 12.34 Threonine 5.72 Leucine 2.80 Tryptophane 2.80 Proline 1.52 Isoleucine 0.60 Valine 0.54 Alanine 0.41 Glycine 0.40 Lysine 0.22 Histidine 0.12 Total 94.97 Band II (g/IOOg amino acid) 24.40 5.66 17.63 17.11 17.06 8.47 1.72 0.00 0.00 0.98 0.66 0. 33 0.98 0.00 0.00 95.00 118. antibiotic compounds unique (ie. have they been isolated before) ? During the past thirty years of antibiotic screening, members of the genus Bacillus have proven .to be the most successful of all in the order of Eubacteriales in the exploration for new antibiotics (Meyers et al, 1973). Furthermore, it is generally recognized , these antibiotics are mainly peptide in structure. Shoji (1978) reports that the number of known antibiotics from Bacillus is approximately 117, of which 80 members are peptides. Most of the peptide antibiotics formed by these organisms are composed entirely of amino acids (eg. tyrocidines, gramidicin S) , where as others may contain amino acids plus other constituents (eg. ring structures, amino sugars, fatty acids) (Katz & Demain, 1977). Why would Bacillus AOG produce two antibiotic substances which are relatively similar in amino acid composition? Examination of the literature reveals that in the case of bacilli, this is the rule rather than the exception. Katz & Demain (1977) state that generally a family of closely related peptides rather than a single substance is produced by a , Bacillus organism. The members may differ from each other by one, or at most, a few amino acids (Perlman & Bodanszky, 1971). For example. Bacillus subtilus produces at least fourteen distinct (but related) anti-biotics; B. brevis also produces a multiplicity of peptide antibiotics (23) (eg. edeine, gramicidin, and tyrocidine) (Sadoff, 1972). The antibiotics of the Bacillaceae are similar in the following general prop-erties: 1) they are peptides comprised of 6-15 amino acids; 2) they are produced by cells after exponential growth during the course of 119. s p o r u l a t i o n ; 3) t h e i r d i r e c t s y n t h e s i s does not i n v o l v e mRNA or ribosomes as i n the case of normal protein s y n t h e s i s , and 4) peptide a n t i b i o t i c s s p e c i f i c a l l y i n h i b i t important c e l l u l a r processes (eg. DNA s y n t h e s i s ) . L i t t l e p r o g r e s s has been made in e l u c i d a t i n g the function(s) of a n t i b i o t i c s in the p r o d u c i n g organism (Bu'Lock, 1961). Generally members of the Bacillaceae are c h a r a c t e r i z e d by t h e i r a b i l i t y to produce resistant endospores and in t h i s course, to elaborate peptide a n t i b i o t i c s (Hodgson, 1970). It thus seems appropriate to assume that the s p o r u -lation a n t i b i o t i c s also e x e r t some c o n t r o l l i n g outcome in the cells from which they have been e x c r e t e d . As Bu'Lock (1961) r e p o r t s , a n t i b i o t i c s y n t h e s i s is a means of keeping the c e l l u l a r machinery i n working o r d e r d u r i n g the time when cell growth is not possible due to unfavorable conditions. Hodgson (1970) believes that peptide a n t i b i o t i c s might be used in several ways by an organism such as modifiers of the cell membrane; for example, ion c a r r i e r s , modifying permeability c h a r a c t e r i s t i c s . Furthermore, Sadoff (1972) states that the peptide a n t i b i o t i c s produced by the b a c i l l i e x h i b i t effects upon membrane sy n t h e s i s and "function, cell wall s y n t h e s i s , and nucleic acid s y n t h e s i s . It:was~contended that the a n t i b i o t i c when produced by an organism, may act as selective modifiers of cell f unction -ie. r e p r e s s i n g or i n h i b i t i n g vegetative cell macromolecular s y n t h e s i s . The quantities r e q u i r e d in sporulation might amount to r e l a t i v e l y few molecules per c e l l . Thus an t i b i o t i c production by B a c i l l u s AOG probably represents an amplification of a normal event which occurs to a lesser degree in all 120. aerobic sporulating bacilli. Although the mechanism of antibiotic synthesis varies from protein synthesis, there appears to be a competition between these two processes for the amino acids available in the cell. For example, during active cellular growth of B. licheniformis with high protein synthesis, practically no bacitracin was produced (Studer, 1967). In contrast, bacitracin synthesis was high when the requirement for protein synthesis was inhibited. It thus seems plausible that at thermophilic temperatures Bacillus AOG may require added protein synthesis for thermophilic requirements (eg. to maintain the integrity of the cell components) and thus excess amino acids are not available for production of peptide antibiotics. At lower temperatures (25°C) Bacillus AOG is no longer in a "stressed-state", less cell wall material is needed, and thus there might be a larger number of available amino acids. An abundance of amino acids at mesophilic temperatures might normally result in cell death due to un-balanced growth (Weinberg, 1971). Bacillus AOG may therefore have a system of detoxifying itself by incorporating the "metabolites (ie. amino acids) to antibiotics which are then released from the cell. Hence, the production of antibiotics might be more likely to occur at lower mesophilic temperatures. Matteo et al (1976) reports that it is quite possible that peptide antibiotic formation is controlled by carbon and nitrogen catabolite re-pression or is under growth rate control; manipulations which affect those controls (eg. possibly large fluctuations in temperature) would be expected 121. to modify the temporal rel a t i o n s h i p between an t i b i o t i c s y n t h e s i s and growth. S t i l l under c u r r e n t c o n s i d e r a t i o n , is the p o s s i b i l i t y that an t i b i o t i c s f u n c t i o n to k i l l or i n h i b i t the growth of other organisms i n nature,there-by p r o v i d i n g a competitive advantage to the p r o d u c i n g organism (Go t t l i e b , 1976). One might speculate that Bac i l l u s AOG has evolved to produce an t i b i o t i c s at only mesophilic temperatures' because there is a much l a r g e r number of mesophilic competitive organisms as compared to those at thermophilic temperatures - especially in mushroom compost from where Ba c i l l u s AOG was i n i t i a l l y isolated. The three main classes of a n t i f u n g a l peptides (possessing only s l i g h t a n t i b a c t e r i a l a c t i v i t y ) produced by B a c i l l u s sp. are the bacillomycins, mycobacillins, and f u n g i s t a t i n a n t i b i o t i c s ( S t u d e r , 1967). Similar to the B a c i l l u s AOG i n h i b i t o r , they have proven to be e x t r a c e l l u l a r , acidic ( f u n g i s t a t i n - b a s i c ) , contain no s u l p h u r amino a c i d s , and are heat-stable peptides. They are also soluble in polar, and insoluble in non-polar o r g a n i c solvents (Sharon et a l , 1954).. Most of these a n t i b i o t i c s , although isolated many-years ago are known only in t h e i r q u a l i t a t i v e composition. Except for f u n g i s t a t i n "(which contains u n i d e n t i f i e d amino a c i d s ) , both the bacillomycin and mycobacillin family contain only known amino acids with no other components.-However, closer examination reveals that severe d i s p a r i t i e s o ccur between these B a c i l l u s a n t i b i o t i c classes and the B a c i l l u s AOG compound -and thus can not be grouped into these families. For example, both 122. mycobacillin and bacillomycin are n i n h y d r i n negative (Sharon et a l , 1954), and s i g n i f i c a n t d i f f e rences in content of s p e c i f i c amino acids (eg. b a c i l l o -mycin contains only 5 amino a c i d s ) , U.V. a d s o r p t i o n , and solvent s o l u b i l i t y o c c u r s . A f u r t h e r e x t e n s i v e examination of the l i t e r a t u r e concerning fungal peptide an t i b i o t i c s reveals as of y e t , no known an t i b i o t i c s with id e n t i c a l p r o p e r t i e s (eg. amino a c i d s , U.V. s p e c t r a , solvent s o l u b i l i t y etc.) as that o bserved in thi s thesis in v e s t i g a t i o n of the Ba c i l l u s AOG a n t i b i o t i c . One may reasonably conclude th e r e f o r e , that the i n h i b i t o r produced by Ba c i l l u s AOG is a unique an t i b i o t i c compound and thus has been g i v e n the name Chaetomacin. 123. CONCLUSIONS Mushroom cultivation is now one of the most intensive and most technically demanding of all vegetable cultivations practised throughout the world (Smith, 1969). The fact that basidiomycetes convert waste materials into a highly flavored proteinaceous food is clearly relevant to the requirements of both the emerging and technologically advanced countries. The future role of mushrooms will be governed by the economics of production methods and costs relative to other animal and vegetable foods (Hayes S Nair, 1975). However, escalating costs for maintenance and operating continue to plague growers with the high cost of labor being the most significant (greater than 50% of the total production costs). The commercial mushroom industry has suffered serious crop losses for years by the uncontrollable damage in compost beds caused by the weed mold Chaetomium olivaceum. There is currently no known means to successfully control this pathogen. This thesis investigation resulted in the isolation of a thermophilic Bacillus sp (resembling B. coagulans -resistant to 0.02% sodium azide, acidophilic) which showed dramatic activity against C. olivaceum on TSY agar plates. Studies involving both conventional and hydroponic mushroom cultivation methods demonstrated Bacillus AOG to significantly protect the mushroom from Olive green mold,damage as well as to increase yields of Agaricus bisporus. 124. The observation that initiation and development of fruitbodies were not retarded shows that Bacillus AOG had no apparent inhibitory effect on organisms such as P. putida, known to stimulate the formation of sporophores. These experiments clearly show the benefits resulting from selective protection through controlled fermentation of the nutrient substrate. The isolation of a thermophilic microorganism antagonistic towards Olive green mold is an extremely unique and significant finding when considering microbial development of the commercial mushroom industry. The reserve of chemical controls for mushroom pathogens continues to shrink as various materials become ineffective or are removed from usage because of residues or suspected residues of toxic compounds in the marketed product (Ingratta, 1980). This novel finding (Bacillus AOG) could potentially be used as an effective application of biological control in this solid state fermentation process. Mushroom producers now have the capability of protecting their crops from Chaetomium damage for the first time in the history of this industry. Substrates assume the greatest share of production costs (Hayes & Wright, 1979). The main advantages of hydroponics over composting are: much less labor intensive, more efficient regulation of nutrient composition (ie. completely controlled fermentation with known thermo-philes present), efficient use of water (important in arid countries), protection from pathogens, and permanence of medium (compost must be discarded regularly - whereas vermiculite may last for several years 125. and is v e r y l i g h t to fac i l i t a t e transportation) - pos s i b l y leading to eventual continuous c u l t u r e techniques. Ba c i l l u s AOG was shown to produce a potent an t i b i o t i c (named Chaetomacin) at mesophilic temperatures. E x t r a c t i o n of thi s compound with n-butanol revealed it to be a stable substance, e f f e c t i v e against other fungi and Ba c i l l u s species. P u r i f i c a t i o n t h r o u g h t h i n layer chroma-tography revealed two compounds with close R^ values. Amino acid a n alysis showed these two bands to be similar in s t r u c t u r a l compostion. Examination of the l i t e r a t u r e reveals no other p r e v i o u s l y isolated a n t i b i o t i c s which are identical to the i n h i b i t o r s found in thi s thesis s t u d y . Chaetomacin could potentially be extended into the preparation of d i f f e r e n t i a l media and in the protection of foods and plants from fungal i n v a s i o n . 126. LITERATURE CITED 1. Allen, M.B. 1953. The thermophilic aerobic sporeforming bacteria. Bact. Reviews 17:125-173. 2. Anon. 1981. Good growth potential seen for Canadian mushroom industry. Food in Canada 41.: 15. 3. Atkins, F .C. 1972. Mushroom growing today. Faber and Faber Limited, London. 4. Aulin-Erdtman, G. 1949. Ultraviolet spectroscopy of lignin and lignin derivatives. Tappi 32:160-166. 5. Baker, R. 1968. Mechanisms of biological control of soil-borne pathogens. Ann. Rev. Phytopath. 6:263-294. 6. Baker, K.F. and Cook, R.J. 1974. Biological control of plant pathogens. W.H. Freeman C o . , San Francisco, CA . 7. Baker, K.F. and Snyder, W.C. 1965. Ecology of soil-borne plant pathogens. Univ. of Calif. Press, Berkeley, CA . 8.' Barret, T. 1956. The commercial cultivation of mushrooms. J .E .R . Simons L td . , Harlow, Essex. 9: Beach, W.S. 1937. Control of mushroom diseases and weed fungi. Pennsyl. State College, Penn. Bull.# 351 :l-32. 10. Beardsell, D .T .R . 1979. How we grow mushrooms. Mushr. J . 84:545, 549. 11. Bels-Koning, H . C . , Gerrits, J . P .G . and Vaandrager, M.H. 1962. Interaction between mushroom mycelium and some fungi. Mushr. Sci. 15:165-169. 12. Bels-Koning, H .C . , Gerrits, J . P .G . and Vaandrager, M.H. 1962. Some fungi appearing towards the end of composting. Mushr. Sci 5:165-169. 13. Bi.tner, C.W. 1972. The pathogens of mushroom spawn {Agaricus bisporus). Mushr. Sci. 8:601-606. 127. 14. B l o c k , S.S. 1964. Composting conversion of soli d wastes for mushroom growing. Biotech. Bioeng. 6:403-418. 15. Brancato, F.P. and G o l d i n g , N.S.-1953. The diameter of the mold colony as a reliable measure of growth. Mycol. 45: 848-864. 16. B r a u n s , F.E. 1952. The chemistry of l i g n i n . Academic Press Inc., New Y o r k , N.Y. 17. Brewer, D.W., Jerram, W.A., Meiler, D. and T a y l o r , A. 1970. The t o x i c i t y of c o c h l i o d i n o l , an anti b i o t i c metabolite of Chaetomium spp. Can. J . Micb. 23:845-851 . 18. Brewer, D. and T a y l o r , A. 1978. The production of t o x i c metabolites by Chaetomium spp isolated from soils of permanent pasture. Can. J . Micb. 24:1082-1086. 19. B r i a n , P.W. 1957. The ecological s i g n i f i c a n c e of a n t i b i o t i c p r o d u c t i o n . In "Microbial Ecology", p. 168-188, edited by R.E.O. Williams, and C C . S p i c e r . Cambridge Univ. P r e s s , London. 20. Brown, H.P. 1937. Mushroom bed invaders - t h e i r habits and t h e i r means of c o n t r o l . A g r . Caz. N.S.W. 48; 436-439. 21. Buchanan, R.E. and Gibbons, N.E. (E d . ) . 1975. Bergey's manual of determinative bacteriology. 8th e d i t i o n . The Williams and Wilkins Co., Baltimore, MD. 22. Bu'Lock, J.D. 1961. Intermediary metabolism and a n t i b i o t i c s y n t h e s i s . Adv. A p p l . Micb. 3:293-342. 23. B u r d e n , O.J. and Peterson, R.A. 1972. C u l t i v a t i n g mushrooms. Queensland A g r i c . J . 98:63, 141. 24. C a i l l e u x , R. 1973. Mycoflora du compost destine a la c u l t u r e die champignon de couche. Revue Mycol. 7:14-35. 25. Campbell, L.L. and S n i f f , E.E. 1959. Folic acid requirements of Bacillus coagulans. J . Bact. 78:267-271. 26. Chahal, D.S., Sekhon, A. and Dhaliwal, B.S. 1975. Degradation of wheat straw by the f u n g i isolated from s y n t h e t i c mushroom compost. In "Proc. Int. Biodegradation Symp.", p. 665-671. Edited by J.M. S h a r p l e y , and A.M. Kaplan. A p p l . S c i . P r e s s , England. 128. 27. Chang, S .T . 1980a. Mushrooms as human food. Bioscience 30:399-401. 28. Chang, S .T . 1980b. Mushrooms from waste. Food Policy 5:64-65. 29. Chang, S .T . and Hayes, W.A. 1978. The biology and cultivation of edible mushrooms. Academic Press, NY. 30. Chanter, D.P. and Spencer, D.M. 1974. The importance of thermophilic bacteria in mushroom compost fermentation. Scientia Hort. 2:249-256. 31. Christianson, C M . and Kaufman, H.H. 1969. Grain storage — the role of fungi in quality loss. Univ. of Minneapolis Press. Minneapolis, Minn. 32. Couvy, J . 1972. Etude de I'induction de la fructification chez Agaricus bisporus (lange) Sing [Psailliota hortensis Cke) : action du glucose. Comptes rendus hebdemadaire des seances de I'Academie des Sciences 274. p. 2475-2477. 33. Davis, A . C . 1938. Mushroom pests and their control. Circ . #457, U .S .D.A. 34. Dawson, W.M. 1977. Mushrooms: casing techniques. Agric. North. Ireland 52:160-162. 35. , DoAmaral, J . F . 1964. Diseases complicating the growth of the edible mushroom. Biologico 30:299-301. 36. Doetsch, R.N. 1981. Determinative methods of light microscopy. In "Manual of Methods of General Bacteriology",p. 21-33, edited by P. Gerhardt. A . S .M . Washington, DC. 37. Douglas, J . S . 1976. Advanced guide to hydroponics. Drake Publishers, Inc., NY. 38. Dubois, M., Gilles, K .A . , Hamilton, J . K . , Rebers, P.A. and Smith,F. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350-356. 38. Eastwood, D.J. 1952. The fungus flora of composts. Trans. Brit Mycol. Soc. 35_:215-220. 129. 39. Eddy, B.P. and Jacobs, L. 1976. Mushroom compost as a source of food for Agaricus bisporus. Mushr. J. 38:56-59,67. 40. Eger, C. 1962. Ein fluchtiges stoffwechsel product des kultur-champignons, Agaricus (Psalliota) bisporus (Ige.) Sing, mit antibiotisher wirkung. Natur. Schaften 49:261. 41, Eger, G. 1963. Untersuchungen aur fruchtkorperbildung des kulturchampignons Psalliota bispora Lge. Archiv. fur Mikrob. 39:313-334. 42. Eger, G. 1 972. Experiments and comments on the action of bacteria on sporophore initation in Agaricus bisporus. Mushr. Sci. 8:719-725. 43. Eicker, A. 1977. Thermophilic fungi associated with the cultivation otAgaricus bisporus Lge. Sing. J.S. Afr. Bot. 43:193-207. 44. Eicker, A. 1980. Mesophilic fungi associated with cultivation of Agaricus brunnescens. Trans. Brit. Mycol. Soc. 74:465-470. 45. Fergus, C.L. 1964. Thermophilic and thermotolerant molds and actinomycetes of mushroom compost during peak-heating. Mycol. 56:267-284. 46. Fergus, C.L. 1971. The heat resistance of some thermophilic fungi on mushroom compost. Mycol. 63:675-679. 47. Fergus, C.L. 1978. The fungus flora of compost during mycelium colonization by -the cultivated mushroom Agaricus brunnescens. Mycol. 70:636-644. 48. Fergus, C.L. 1982. The heat resistance of some mesophilic fungi isolated from mushroom compost. Mycol. 74:149-152. 49. Fermor, T.R., Smith, J.F. and Spencer, D.M. 1979. The micro-flora of experimental mushroom composts. J. Hort. Sci. 54:137-147. 50. Fletcher, J.T. 1979. Bacteria and mushrooms. Mushr. J. 82:45I-457. 51. Fordyce, C. 1970. Relative numbers of certain microbial groups present in compost used for mushroom (Agaricus bisporus) propagation. Appl. Micb. 20:196-199. 130. 52. G a r r e t t , S.D. 1955. A c e n t u r y of root-disease i n v e s t i g a t i o n s . Ann. A p p l . B i o l . 42:211-219. 53. G a r r e t t , S.D. 1956. Biology of ro o t - i n f e c t i n g f u n g i . Cambridge, Un i v . Press, Boston, MASS. 54. G a r r e t t , S.D. 1965. Toward biological control of soil-borne plant pathogens. In "Ecology of Soil-borne Plant Pathogens", p. 4-17, edited by K.F. Baker and W.C. Sny d e r , Univ. of C a l i f . P r e s s , B e r k e l e y , CA. 55. G e r r i t s , J.P.G. 1968. Organic compost constituents and water u t i l i z e d by the c u l t i v a t e d mushroom d u r i n g spawn r un and cr o p p i n g . Mushr. S c i . 7:111-126. 56. G e r r i t s , J.P.G. 1977. The si g n i f i c a n c e of gypsum applied to mushroom compost in p a r t i c u l a r i n relation to the ammonia content. Neth. J . A g r i c . S c i . 25:288-302. 57. G e r r i t s , J.P.G. 1980. Hoe lang d u u r t uitzweten i n een tunnel en wat is de beste temperatuur? Champignoc. 24: 49-59. 58. G e r r i t s , J.P.G., B e l s - K o n i n g , J.C. and Mu l l e r , F.M. 1965. Changes i n compost constituents d u r i n g composting, p a s t e u r i z a t i o n , and c r o p p i n g . Mushr. S c i . 6:225-243. 59. G i n d r a t , D. 1979. Biocontrol of plant diseases by inoculation of fr e s h wounds, seeds, and soil with antagonists. In " S o i l -borne Plant Pathogens", p. 541-551, edited by B. S c h i p p e r s and W. Gams. Academic P r e s s , N.Y. 60. Gordon, R.E., Haynes, W.C. and Pang, C H . 1973. The genus Bacillus. A g r i c u l t u r e Handbook No. 427. A g r i c . Res. S e r v . U.S.D.A., Washington, D.C. 61. G o t t l i e b , D. 1976. The prod u c t i o n and role of anti b i o t i c s i n s o i l . J . A n t i b . 29:987-1000. 62. Hammer, B.W. 1915. Bacteriological studies on the coagulation of evaporated milk. Iowa A g r i c . Exp. Stat. B u l l . 1 9:119-1 31. 63. Han, Y.S., S h i n , K.C. and Kim, D.S. 1974. Some biological studies of Mycogone perniciosa Magn. causing wet bubble in c u l t i v a t e d mushroom Agaricus bisporus (Lange) S i n g . Korean J . Mycol. 2:7-14. 131. 64. Hatch, R.T. and F i n g e r , S.M. 1979. Mushroom fermentation. In "Microbial Technology", p. 179-199, 2nd ed.. Vol II. Edited by H.J. Peppier and D. Perlman. Academic P r e s s , N.Y. 65. Hayes, W.A. 1969. Microbiological changes in composting wheat straw/horse manure mixtures. Mushr. S c i . 7:173-186. 66. Hayes, W.A. 1970. Fumigation - its application to commerical mushroom growing. MCA B u l l . 257:21 3. 67. Hayes, W.A. 1972. N u t r i t i o n a l factors i n relation to mushroom pro d u c t i o n . Mushr. S c i . 8:663-674. 68. Hayes, W.A. 1974a. Mushroom c u l t i v a t i o n - prospects and developments. Proc. Bioc. 9:21-24, 28. 69. Hayes, W.A. 1974b. Microbiological a c t i v i t y i n the casing layer and it s relation to p r o d u c t i v i t y and disease c o n t r o l . In "The C a s i n g Layer", p. 27-48, edited by W.A. Hayes. London. 70. Hayes, W.A. 1977. Mushroom n u t r i t i o n and the role of micro-organisms i n composting. In "Composting", p. 1-20, edited by W.A. Hayes. London. 71. Hayes, W.A. and N a i r , N.C, 1974. Effects of vo l a t i l e metabolic b y - p r o d u c t s of mushroom mycelium on the ecology of the casing l a y e r . Mushr. S c i . 9:259-268. 72. Hayes, W.A. and N a i r , N.C. 1975. The c u l t i v a t i o n of Agaricus bisporus and other edible mushrooms. In " The Filamentous F u n g i " , p. 212-248, Vol I, edited by J.E. Smith and D.R. B e r r y . 73. Hayes, W.A. and Randle, P.E. 1969a. Use of molasses as an ing r e d i e n t of wheat straw mixtures used for the preparation of mushroom composts. Report Glasshouse C r o p s Res. Inst. 142-147. 74. Hayes, W.A. and Randle, P.E. 1969b. The use of water soluble c a r b o hydrates and methyl bromide in the preparation of mushroom compost. MCA B u l l . 28:81-97. 132. 75. Hayes, W.A. and Randle, P.E. 1970. A n alt e r n a t i v e method of pr e p a r i n g compost using methyl bromide as a p a s t e u r i z i n g agent. Report Glasshouse Crops Res. Inst. p. 166-169. 76. Hayes, W.A., Randle, P.E. and Last, F.T. 1969. The nature of the microbial stimulus a f f e c t i n g sporophore formation i n Agaricus bisporus (Lge.) S i n g . Ann. A p p l . B i o l . 64:177-187. 77. Hayes, W.A. and Wright, S.H. 1979. Edible mushrooms. In "Economic Microbiology", p. 141-176, edited by A.H. Rose. Academic P r e s s , N.Y. 78. Henis, Y. and Chet, I. 1975. Microbiological control of plant pathogens. In "Advances i n A p p l i e d Microbiology", V o l . 1_9, p. 85-111, edited by D. Perlman. Academic P r e s s , N.Y. 79. Henssen, A. 1957. Uber die bedeutung der thermophilem mikroorganismen f u r die ze r s e t z u n g des stallmistes. A r c h . Mikrob. 27:63-81. 80. Hodgson, B. 1970. Possible roles for ant i b i o t i c s and other biologically active peptides at sp e c i f i c stages d u r i n g sporulation of Bacillaceae. J . Theor. B i o l . _30_: 111; 119. 81. Hudson, K.E. 1972. Nematodes as biological control agents: t h e i r possible application in c o n t r o l l i n g insect pests of mushroom cr o p s . Mushr. S c i . 8:193-197. 82. Huhnke, W. 1970. Modern mushroom farming. S c i . J . 6:62-66. 83. Huhnke, W. and VonSengbush, R. 1968. Champignonanbau auf i nicht kompostiertem n'ahrsubstrat. Mushr. S c i . 7:405-409. 84. Humphreys, T.W. and Costilow, R.W. 1957. Observations on the n u t r i t i o n a l requirements of Bacillus coagulans. Can. J . Micb. 3:533-541. 85. Hussey, N.W. 1969. Biological control of mushroom pests - fact or fantasy. MGA. B u l l 238:448-465. 86. Hussey, N.W. 1972.' Pests in p e r s p e c t i v e . Mushr. Sci 8:183-192. 87. I n g r a t t a , F. 1980. Where are we going? Mushr. J . 85:47-51. 133. 88. Ingratta, F. 1980. Mushrooms - mystery, magic, or science? Highlights Agic. Res. Ont. 3:8-10. 89. Ivanovich-Biserka, B. 1972. Dealing with microbiological trouble-makers in commercial spawn production of Agaricus bisporus, L. Mushr. Sci. 8:305-314. 90. Jackson, R.M. 1 965. Antibiosis and fungistasis of soil micro-organisms. In "Ecology of Soil-borne Plant Pathogens", p. 363-373, edited by K.F. Baker and W.C. Snyder, Univ. of Calif. Press, Berkeley, CA . 91 Johnson, D.B. , Moore, W.E. and Zank. L .C . 1961. The spectrophotometric determination of lignin in small wood samples. Tappi 44:793-798. 92. Kanwar, Z .S . , Khanna, P.K. and Tharya, M.L. 1979. Seed mycoflora of sunflower and their control. Pest. 13: 41-44. 93. Katz, E. and Demain, A . 1977. The petide antibiotics of Bacillus: chemistry, biogenesis, and possible functions. Bact. Rev. 41_: 449-474. 94. Kinrus, A . 1974. Techniques and methods in the U.S. mushroom industry. Mushr. Sci. 9:165-173. 95. Kleyn, J . C , Johnson, W.M. and Wetzler, T . F . 1981. Microbial aerosols and actinomycetes in etiological considerations of mushroom worker's lungs. Appl . Env. Micb. 41:1 454-1460. 96. Kleyn, J . G . and Wetzler, T . F . 1981. The microbiology of spent mushroom compost and its dust. Can. J . Micb. 27:748-753. 97. Kneebone, L.R. and Merek, E.L. 1959. Brief outline of and control for mushroom pathogens and weed moulds, indicator moulds and competitors. MGA. Bull. 113:146-152. 98. Krieg, N.R. and Gerhardt, P. 1981. Solid culture. In "Manual of Methods for General Bacteriology", p. 143-150, edited by P. Gerhardt. A . S . M . , Washington, D .C. 99. Kurtzman, R.H. 1979. Mushrooms: single cell protein from cellulose. In "Annual Reports on Fermentation Processes", p. 305-339, Vol. 3, edited by D. Perlman. Academic Press, N.Y. 134. 100. Laborde, J . and Delmas, J . 1969. Preparation express des substrats. Bull . FNSACC. JJ): 2093-2109. 101. Laborde, J . , Delmas, J . and d'Hardemare. 1968. Note preliminaire sur quelques aspects de I'equilibre microbiologique des composts. Mushr. Sci. 7:187-203. 102. Laborde, J . , Delmas, J . , Lamau, J . L . and Berthaud, J . 1972. La preparation express des substrats (P.E.S.) pour la culture du champignon de couche. Mushr. Sci 8:675-706. 103. Lambert, E.B. 1938. Principles and problems of mushroom culture. Bot. Rev. 4:397-426. 104. Lambert, E.B. and Ayers, T . T . 1953. Diseases of the common mushroom. U .S .D .A . Yearbook p. 478-482, Washington, D.C. 105. . LaTouche, C . J . 1949. Fungi found in composts and mushroom beds. Mush. Res. Stat. Rep. 48:64. 106. Lin, C .Y . and Chen, D.W. 1977. Actinomycetes as biological control agents for calve's brain fungus Diehliomyces micro-sporus in mushroom culture. Natn. Chung Hsing Univ. Taichung, Taiwan, p. 125-143. 107. L iu , T . Y . and Chang, Y . H . 1971. Hydrolysis of proteins with p-Toluenesulfonic acid. J . Biol. Chem. 246:2842-2848. 108. Long, P.E. and Jacobs, L. 1968. Some observations on CG*2 and sporophore initiation in the cultivated mushroom. Mushr. SciL 7:373-383. 109. Long, P.E. and Jacobs, L. 1974. Aseptic fruiting of the cultivated mushroom, Agaricus bisporus. Trans. Brit. Mycol. Soc. 63:99-107. 110. Loudon, J . C . 1850. An encyclopedia of gardening; comprising the theory and practice of horticulture, floriculture, aboriculture and landscape gardening. 2nd ed. Longman, London. 111. Marshall, R. and Beers, R.J. 1967. Growth of Bacillus coagulans in chemically defined media. J . Bact. 94:517-521. 135. 112. Matteo, C.C., Cooney, C L . and Demain, A.L. 1976. Production of gramicidin S synthetase by Bacillus brevis i n continous c u l t u r e . J . Gen. Micb. 96:415-422. 113. Meyers, E., Brown, W., P r i n c i p e , P., Rathnum, M. and P a r k e r , W. 1973. EM 49, a new peptide a n t i b i o t i c . J . A n t i b . 26:444-448. 114. M i n i s t r y of A g r i c u l t u r e . 1980. Mushroom production guide. V i c t o r i a , B.C. 115. M u l l e r , F.M. 1 965. Some thoughts about composting. Mushr. Sci 6:213-222. 116. N a i r , N . C 1974. Methods of control for ba c t e r i a l blotch disease of the c u l t i v a t e d mushroom with s p e c i f i c reference to biological c o n t r o l . Mushr. J . 16:140. 117. N a i r , N.G. 1980. Weed-moulds in mushroom c u l t i v a t i o n . A g r . Gaz. N.S.W. 91:36-69. 118. N a i r , N . C and Fahy, P .C 1972a. Bacteria antagonistic to Pseudomonas tolaasii and t h e i r control of brown blotch of the c u l t i v a t e d mushroom Agaricus bisporus. J . A p p l . Bact. 35:439-442. 119. N a i r , N.G. and Fahy, P .C 1972b. Prospects for biological control of brown b l o t c h . MGA. B u l l . 270:252. 120. N a i r , N.G. and Fahy, P . C 1976. Commercial application of biological c ontrol of mushroom bacte r i a l b l o t c h . A u s t . J . A g r i c . Res. 27:415-422. 121. N a i r , N.G. and Hayes, W.A. 1974. Suggested role of carbon dioxide and oxygen in the casing s o i l . In "The Casi n g L a y e r " , edited by W.A. Hayes, MGA, England. 122. N a i r , N . C , S h o r t , C C and Hayes, W.A. 1974. Studies on the gaseous environment of the casing l a y e r . Mushr. S c i . 9_:245-257. 123. N i k , W. 1980. Seed-borne fu n g i of soybean and t h e i r c o n t r o l . P e r t . 3:125-132. 124. O'Donoghue, D. 1965. Relationship between some compost factors and t h e i r effects on the y i e l d of A g a r i c u s . Mushr. S c i . 6:245. 136. 125. O l i v i e r , J.M. and Cuillaumes, J . 1976. Effect antagoniste exerce i n v i t r o par Ie mycelium de Psalliota bispora Lange v i s - a - v i s de dif f e r e n t e s especes fongiques et bacteriennes. Ann. Phytop. 8:213-231. 126. P a r k , D. 1960. Antagonism - the background of soil f u n g i . In "The Ecology of Soil F u n g i " , p. 148-159, edited by D. Parki n s o n and J.S. Wald. Live r p o o l Univ. P r e s s , L i v e r p o o l , Eng. 127. P a r k , J.Y. 1971. A stimulative effect of Streptomyces thermovul-garis on the mycelium growth of Agaricus bisporus. MCA. B u l l . 254:78-83. 128. Park , J.Y. and A g n i h o t r i , V.P. 1969. Bact e r i a l metabolites t r i g g e r sporophore formation in Agaricus bisporus. Nature 222: 984. 129. Perlman, D. and Bodanszky, M. 1965. S t r u c t u r a l relationships among peptide a n t i b i o t i c s . Antimicrob. Agent Chemo. 1 965:122-131. 130. Perlman, D. and Bodanszky, M. 1971. Bi o s y n t h e s i s of peptide a n t i b i o t i c s . A n n . Rev. Bioc. 40:449-464. 131. Pope, S., Knaust, H. and Knaust, K. 1962. Production of compost by thermophilic f u n g i . Mushr. S c i . 5:123-126. 132. Potter, C. 1980. New cannery doubles F r a s e r Valley's mushroom capacity. Food in Can. 40:17-1 9. • 133. Renoux-Blondeau, H. 1959. Etude de cert a i n s actinomycetes se develloppart au cours de la pas t e u r i z a t i o n d u i fumir. Leur action s u r le developpement u l t e r i u r du champignon de couche. Mushr. S c i . 4:153-175. 134. Resh, KM. 1978. Hydroponic food p r o d u c t i o n . Woodbridge P r e s s , Santa B a r b e r a , CA. 135. Rettew, R. 1948. Manual of mushroom c u l t u r e . 4th ed., Mushr. S u p p l y Co., Toughkenamon, PA. 136. R i c h a r d s o n , P.N. 1981. Nematodes and biological control of p h o r i d s . Mushr. J . 98:66-69, 71. 137. Ross, R.C. 1976. A s t u d y of the preparation of compost for the c u l t i v a t e d mushroom. PhD. T h e s i s , U n i v e r s i t y of Reading. 137. 138. Ross, R.C. and Harris, P.J. 1982. Some factors involved in phase II of mushroom compost preparation. Scientia Hort. V7:223-229. 139. Royse, J . and Schisler, L .C . 1980. Mushrooms: their consumption, production, and culture development. Interdis. Sci. Rev. 5:324-332. 140. Sadoff, H.L. 1972. Sporulation antibiotics of Bacillus species. In "Spores V " , p. 157-166, edited by H.O. Halvorson, R. Hanson, L.L. Campbell. A . S . M . , Washington, D .C . 141. San Antonio, J .P . 1966. Effects of injection of nutrient solutions into compost on the yield of mushrooms (Agaricus bisporus). Proc. Amer. Soc. Hort. Sci. 89:415-422. 142. Sankov, E .A. , Suchkova, C C . and Andreeva, K.I. 1972. Biological damage to wool fibers as determined by dyeing. Izv. Vyssh. Vcheb. Zaved. Tekhnol. Tekst. Prom. 4:154-155. 143. Schisler, L .C . 1980. Composting. Mushr. News. 2:5-13. 144. Schroth, M.N. and Hancock, J . C 1981. Selected topics in biological control. In "Annual Reviews of Microbiology", p. 453-476, Vol. 35, edited by M.P. Starr, J . L . Ingraham, and A. Balows. Palo Alto, CA . 145. Sekita, S., Yoshihara, K., Natori, S. , Udayawa, Muroi, T . , Sugiyama, Y . , Kurata, H. and Umeda, M. 1981. Mycotoxin production by Chaetomium spp. and related fungi. Can. J . Micb. 27:766-772.. 146. Sharon, N. , Pinsky, A . , Turner-Graff, R. and Babad, J . 1954. Classification of the antifungal antibiotics from Bacillus subtilus. Nature 174:1190-1191. 147. Shoji, J . 1978. Recent chemical studies on peptide antibiotics from the genus Bacillus. In " Advances in Applied Micro-biology", p. 187-214, Vol. 24, edited by D. Perlman. Academic Press, N.Y. 148. Sinden, J.W. 1971. Ecological control of pathogens and weed-molds in mushroom culture. In "Annual Reviews of Phytopathology", p. 411-432, Vol. 9, edited by J . G . Horsfall, K.F. Baker, and G.A. Zentmyer. Palo Alto, CA . 138. 149. Sinden, J.W. 1972. Disease problems in technologically advanced mushroom nurseries. Mushr. Sci. 8:126-130. 150. Sinden, J.W. and Hauser, E. 1953. The nature of the composting process and its relation to short composting. Mushr. Sci. 2: 123-130. 151. Smibert, R.M. and Krieg, N.R. 1981. General characterization. In "Manual of Methods for General Bacteriology", p. 409-443, edited by P. Gerhardt. A . S . M . , Washington, D .C . 152. Smith, J . 1969. Commercial mushroom production. Proc. Bioc. 4:43-46, 52. 153. Smith, J . F . 1974. Selective substrates and rapid methods of preparation. Mushr. J . 23:424-426. 154. Smith, N.R., Gordon, R.E. and Clark, F.E. 1952. Aerobic spore-forming bacteria. U .S .D.A. Monograph #16, Washington, D .C . 155. Stahl, E. 1969. Thin layer chromatography. 2nd edition, Springer-Verlag, 156. Stanek, M. 1959. The germination of the basidiospores of cultivated mushroom - A. bisporus. The volatile stimulant of germination, produced by mycelium of A. bisporus. Ces. Mykol . JJ: 241-251. 157. Stanek, M. 1967a. Microorganisms colonizing mushroom compost during fermentation. Mykologicky sbornik 4:57-63. 158. Stanek, M. 1967b. Nutrient substrate of microorganisms of mushroom compost. Folia Micb. 12:397. 159. Stanek, M. 1968. Die wirkung der zellulosezersetzenden mikroorganismen auf das wachstum des champignons. Mushr. Sci. 7:161-172. 160. Stanek, M. 1972. Microorganisms inhabiting mushroom compost during fermentation. Mushr. Sci 8_:797-811. 161. Stanek, M. 1974. Bacteria associated with mushroom mycelium Agaricus bisporus (Lge.) Sing, in hyphosphere. Mushr. Sci. 9:197. 139. 162. Stanek, M. and Rysava-Zatecka, J . 1970. Application of selected strains of thermophilic microorganisms for the fermentation of sterilized synthetic mushroom compost. Mykol. Sbor. 7:103-109. 163. Studer, R.O. 1967. Polypeptide antibiotics of medicinal interest. In "Progress in Medicinal Chemistry", p. 1-58, Vol . 5, edited by C P . Ellis and C .B . West. London, Butterworths. 164. Takeyoshi, A , , Komagata, K., Yoshimura, I. and Mitsugi, K. 1971. Deterioration of synthetic resins by fungi. Hakko. Kogaku. Zasshi. 49:188-194. 165. Tendler, M.D. and Burkholder, P.R. 1961. Studies on the thermophilic actinomycetes. I. Methods of cultivation. Appl . Micb. 9:394-399. 166. T i l l , O. 1962. Champignonkultur auf steriliziertem nahrsubstrat und die wiederverwendung von abgetragenem kompost. Mushr. Sci. 5:127-133. 167. Toleman, E.E. 1969. Mushroom growing for market is specialized business. N.Z. J . Agric . 119:29. 168. Townsley, P.M. 1974. Pure cultural industrial fermentation in the production of mushrooms. Can. Inst. Food Sci. Tech. J . 7:254-255. 169. Treschow, C . 1944. Nutrition of the cultivated mushroom. Dansk. Bot. Ark iv . 6:1-180. 170. Trigiano, R.N. and Fergus, C L . 1979. Extracellular enzymes of some fungi associated with mushroom culture. Mycol. 71:908-917. 171. Trogoff, H. and Ricard, J . L . 1976. Biological control of Verticillium malthousei by Trichoderma viridie spray on casing soil in commercial mushroom production. Plant Dis. Rept. 60:677-680. 172. Turner , R.A. 1956. Chemical studies on bacillomycin. Arch . Bioch. Biophy. 60:364-372. 140. 173. Turner , E.M. 1977. Enzyme activity of Agaricus bisporus in compost. In "Composting", p. 21-24, edited by W.A. Hayes. University of Aston, Birmingham. 174. Urayama, T.1961. Stimulative effect of certain specific bacteria upon mycelial growth and fruitbody formation of Agaricus bisporus (Lge.) Sing. Bot. Mag. Tokyo 74:56-59. 175. Vedder, P.J.C. 1964. Diseases and pests of mushrooms. Croet. Fruit 19:33 176. Vedder, P.J.C. 1978. Modern mushroom growing. Hauser Champignonkulturen A . C . , Cossau-Zurich, Switzerland. 177. Waksman, S .A. 1952. Soil microbiology. Wiley Press, N.Y. 178. Waksman, S.A. and Cordon, T . C . 1939. Thermophilic decompo-sition of plant residues in composts by pure and mixed cultures of microorganisms. Soil Sci. 47:217-224. 179. Waksman, S .A . , Cordon, T . C . and Hulpoi , E.N. 1939b. Influence of temperature upon the microbiological population and decomposition processes in composts of stable manure. Soil Sci. 47:83-112. 180. Waksman, S .A. and Iyer, K.R.M. 1932a. Contribution to our knowledge of the chemical nature and origin of humus: 1. On the synthesis of the "humus nucleus". Soil Sci. 34: 43-70. 181. Waksman, S .A. and Iyer, K.R.M. 1932b. Contribution to our knowledge of the chemical nature and origin of humus: 2. The influence of 'synthesized' humus compounds and of 'hatural humus" upon soil microbiological processes. Soil Sci. 34:71-79. 182. Waksman, S .A. and McCrath, J .M . 1931. Preliminary study of chemical processes involved in the decomposition of manure by Agaricus campestris. Amer. J . Bot. 18:573-581. 183. Waksman, S .A. and Nissen, W. 1931. Lignin as a nutrient for the cultivated mushroom, Agaricus campestris. Sci. 74:271. 184. Waksman, S.A. and Nissen, W. 1932a. Mushroom nutrition: a group of problems in microbiology. Jour. Bact. 23:81-82. 141. 185. Waksman, S.A. and Nissen, W. 1932b. On the nutrition of the cultivated mushroom and the chemical changes brought about by this organism in the manure compost. Amer. J . Bot. 19:514-537. 186. Waksman, S.A., Umbreit, W.W. and Cordon, T . C . 1939c. Thermophilic actinomycetes and fungi in ;sbils and in composts*. Soil Sci. 47: 37-60. 187. Weinberg, E.D. 1971. Secondary metabolism: raison d'etre. Perspect. Biol. Med. 14 = 565-577. 188. Wolf, J . and Barker, A.N. ' 1968. The genus Bacillus: aids to the identification of its species. In "Identification Methods for Microbiologists", p. 93, edited by B.M. Cibbs and D.A. Shapter. Academic Press, N.Y. 189. Wolf, J . and Sharp, R.J. 1981. Taxonomic and related aspects of thermophiles within the genus Bacillus. In "The Aerobic Endospore-forming Bacteria", p. 251-296, edited by R.C.W. Berkely. Academic Press, N.Y. 190. Wood, D.A. 1976. Primordium formation in axenic cultures of Agaricus bisporus (Lge.) Sing. J . Gen. Micb. 95:313-323. APPEND ICES APPENDIX A Statistical analysis of mycelial diameters. CONVENTIONAL LINE 1 : NOTH LINE 2 : BAC DAY DAY STANDARD ERROR INDIVIDUAL SIMREG LINES OF ESTIMATE 1 A = 0.34857E+00 B= 0.18393E+00 SEY=0.6594E-01 2 A= 0.25714E+00 B= 0.27143E+00 SEY=0.1038E+00 SIMREG FOR POOLED DATA 3 A= 0.30286E+00 B= 0.22768E+00 SEY=0.1864E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.403E+00 TEST FOR SLOPES F, 1/ 10.= 0.142E+02 TEST FOR LEVELS F, 1/ 11.= 0.141E+02 OVERALL TEST F, 2./ 10.= 0.226E+02 CORRELATION DEGREES OF COEFFICIENT FREEDOM R= 0.9887 DF= 6. R= 0.9872 DF= 6. R= 0.9351 DF= 13. NOTH LINE 2 : B+C DAY DAY STANDARD ERROR CORRELATION DEGREES OF INDIVIDUAL SIMREG LINES OF ESTIMATE COEFFICIENT FREEDOM A= 0.34857E+00 B= 0.18393E+00 SEY=0.6594E-01 R= 0.9887 DF= 6. A= O.eOOOOE-01 B= 0.28071E+00 SEY=0.1104E+O0 R= 0.9865 DF= S. SIMREG FOR POOLED DATA A= O.20429E+0O B= 0.23232E+00 SEY=0.1437E+00 R= 0.9614 DF= 13. COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.357E+00 TEST FOR SLOPES F. 1/ 10.= O.159E+02 TEST FOR LEVELS F, 1/ 11.= 0.175E+01 OVERALL TEST F, 2./ 10.= 0.999E+01 CONVENTIONAL LINE 1 : NOTH LINE 2 : CHAE DAY DAY STANDARD ERROR INDIVIDUAL SIMREG LINES OF ESTIMATE 1 A= 0.34857E+00 B= 0.18393E+00 SEY=0.G594E-01 2 A= 0.45714E+00 B= 0.14286E+00 SEY=0.6761E-01 SIMREG FOR POOLED DATA 3 A= 0.40286E+00 B= 0.16339E+00 SEY=0.8118E-01 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F. 5./ 5.= 0.951E+00 TEST FOR SLOPES F. 1/ 10.= 0.530E+01 TEST FOR LEVELS F, 1/ 11.= 0.175E+01 OVERALL TEST F, 2./ 10.= 0.387E+01 CORRELATION COEFFICIENT DEGREES OF FREEDOM R= 0.9887 R= 0.9806 R= 0.9746 DF= 6. DF= 6. DF= 13. CONVENTIONAL LINE 1 BAC DAY LINE 2 B+C DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE A= 0.25714E+00 A= O.GOCOOE-01 B= 0.27143E+00 B= 0.28071E+OO SIMREG FOR POOLED DATA A= 0.15857E+00 B= O.27607E+0O SEY=0.1038E+O0 SEY=0.1104E+00 SEY=0.1309E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.884E+00 TEST FOR SLOPES F, 1/ 10.= 0.105E+00 TEST FOR LEVELS F, 1/ 11.= 0.849E+01 OVERALL TEST F, 2./ 10.= 0.395E+01 CORRELATION DEGREES OF COEFFICIENT FREEDOM R= 0.9872 DF= 6. R= 0.9865 DF= 6. R= 0.9767 DF = 13. CONVENTIONAL LINE 1 BAC DAY LINE 2 CHAE DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE A= 0.25714E+00 A= 0.45714E+00 B= 0.27143E+00 B= 0.14286E+00 SEY=0.1038E+00 SEY=0.6761E-01 SIMREG FOR POOLED DATA A= O.35714E+O0 B= 0.20714E+O0 SEY=0.2334E+0O COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.236E+01 TEST FOR SLOPES F, 1/ 10.= 0.302E+02 TEST FOR LEVELS F, 1/ 11.= 0.123E+02 OVERALL TEST F, 2./ 10.= 0.376E+02 CORRELATION DEGREES OF COEFFICIENT FREEDOM R= 0.9872 DF= 6. R= 0.9806 DF= 6. R= 0.8866 DF= 13. CONVENTIONAL LINE 1 B+C DAY LINE 2 CHAE DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE A= 0.6CO00E-01 A= 0.45714E+00 B= 0.28071E+00 B= O.14286E+00 SEY=0.1104E+00 SEY=0.6761E-01 SIMREG FOR POOLED DATA A= 0.25857E+00 B= 0.21179E+00 SEY=0.190OE+0O COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F. 5./ 5.= 0.267E+01 TEST FOR SLOPES F, 1/ 10.= 0.318E+02 TEST FOR LEVELS F, 1/ 11.= 0.262E+01 OVERALL TEST F, 2./ 10.= 0.208E+02 THERE ARE 5 VARIABLES AND 6 PAIRS OF LINES (F3.0.4F6.2) CORRELATION DEGREES COEFFICIENT FREEDOM R= 0.9865 DF= 6. R= 0.9806 DF= 6. R= 0.9235 DF= 13. CONVENTIONAL LINE 1 1 2 3 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.403E+00 TEST FOR SLOPES F, 1/ 10.= 0.142E+02 TEST FOR LEVELS F, 1/ 11.= O.141E+02 OVERALL TEST F, 2./ 10.= 0.226E+02 NOTH LINE 2 DAY BAC DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE A= 0.34857E+00 A= 0.25714E+OQ B= 0.18393E+00 B= 0.27143E+QO SIMREG FOR POOLED DATA A= 0.30286E+00 B= 0.22768E+00 SEY=0.G594E-01 SEY=0.1038E+O0 SEY=0.1864E+O0 CORRELATION COEFFICIENT DEGREES OF FREEDOM R= 0.9887 R= 0.9872 R= 0.9351 DF= 6. DF= 6. DF= 13. CONVENTIONAL LINE 1 : NOTH LINE 2 : B+C DAY DAY STANDARD ERROR INDIVIDUAL SIMREG LINES OF ESTIMATE 1 A= 0.34857E+00 B= O.18393E+00 SEY=0.6594E-01 2 A= 0.60OOOE-01 B= 0.28071E+OO SEY=0.1104E+00 SIMREG FOR POOLED DATA 3 A= 0.20429E+00 B= 0.23232E+0O SEY=0.1437E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F. 5./ 5.= 0.357E+CO TEST FOR SLOPES F. 1/ 10.= 0.159E+02 TEST FOR LEVELS F, 1/ 1 1 .= 0.175E+01 OVERALL TEST F. 2.1 10.= 0.999E+01 CORRELATION COEFFICIENT DEGREES OF FREEDOM R= 0.9887 R= 0.9865 R= 0.9614 DF= 6. DF= 6. DF= 13. CONVENTIONAL LINE 1 : NOTH DAY LINE 2 CHAE DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE A= 0.34857E+00 A= 0.45714E+00 B= O.18393E+00 B= O.14286E+OQ SEY=0.6594E-01 SEY=0.6761E-01 SIMREG FOR POOLED DATA A= 0.40286E+00 B= 0.16339E+00 SEY=0.8118E-01 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.951E+OO TEST FOR SLOPES F, 1/ 10.= 0.530E+01 TEST FOR LEVELS F, 1/ 11.= O.175E+01 OVERALL TEST F, 2./ 10.= 0.387E+01 CORRELATION COEFFICIENT DEGREES OF FREEDOM R= 0.9887 R= 0.9806 R= 0.9746 DF= 6. DF= 6. DF= 13. CONVENTIONAL LINE 1 BAC DAY LINE 2 B+C DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE A= 0.25714E+00 A= 0.6000OE-01 B= 0.27143E+00 B= O.28O71E+0O SIMREG FOR POOLED DATA A= 0.15857E+O0 B= 0.27607E+00 SEY=0.1038E+00 SEY=0.1104E+00 SEY=0.1309E+OQ COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.884E+00 TEST FOR SLOPES F, 1/ 10.= O.105E+00 TEST FOR LEVELS F, 1/ 11.= 0.849E+01 OVERALL TEST F. 2./ 10.= 0.395E+01 CORRELATION DEGREES OF COEFFICIENT FREEDOM R= 0.9872 DF= G. R= 0.9865 DF= 6. R= 0.9767 DF = 13. CONVENTIONAL LINE 1 BAC DAY LINE 2 CHAE DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE A= 0.25714E+00 A= 0.45714E+00 B= 0.27143E+00 B= O.14286E+00 SIMREG FOR POOLED DATA A= 0.35714E+00 B= 0.20714E+00 SEY=0.1038E+00 SEY=0.6761E-01 SEY=0.2334E+0O COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.236E+01 TEST FOR SLOPES F, 1/ 10.= 0.302E+02 TEST FOR LEVELS F, 1/ 11.= 0.123E+02 OVERALL TEST F. 2./ 10.= 0.37SE+02 CORRELATION DEGREES COEFFICIENT FREEDOM R= 0.9872 DF= 6. R= 0.9806 DF= 6. R= 0.8866 DF= 13. CONVENTIONAL LINE 1 : B+C LINE 2 : CHAE DAY DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE 1 A= 0.60000E-01 2 A= 0.45714E+O0 B= 0.28071E+OO B= O.14286E+0O SEY=0.1104E+00 SEY=0.6761E-01 SIMREG FOR POOLED DATA A= 0.25857E+O0 B= 0.21179E+00 SEY=0.1900E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.267E+01 TEST FOR SLOPES F. 1/ 10.= 0.318E+02 TEST FOR LEVELS F. 1/ 11.= 0.262E+01 OVERALL TEST F, 2./ 10.= 0.208E+02 CORRELATION DEGREES OF COEFFICIENT FREEDOM R= 0.9865 DF= 6. R= 0.9806 DF= 6. R= 0.9235 DF= 13. TWO PERCENT MALT LINE 1 NOTH DAY BAC DAY LINE 2 INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE A= 0.12143E+00 A = 0.22286E+00 B= 0.23250E+00 B= 0.24536E+00 SEY=0.1052E+00 SEY=0.1008E+00 SIMREG FOR POOLED DATA A= 0.17214E+00 B= 0.23893E+00 SEY=0.1259E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= O.109E+01 TEST FOR SLOPES F, 1/ 10.= 0.218E+0O TEST FOR LEVELS F. 1/ 11.= 0.830E+01 OVERALL TEST F, 2./ 10.= 0.396E+01 CORRELATION COEFFICIENT DEGREES OF FREEDOM R= 0.9822 R= 0.9853 R= 0.9715 DF= 6. DF= 6. DF= 13. TWO PERCENT MALT LINE 1 : NOTH LINE 2 : B+C DAY DAY STANDARD ERROR INDIVIDUAL SIMREG LINES OF ESTIMATE 1 A= 0.12143E+00 B= 0.23250E+00 SEY=0.1052E+00 2 A= 0.26857E+00 B= 0.21643E+00 SEY=0.8924E-01 SIMREG FOR POOLED DATA 3 A= 0.1950OE+0O B= 0.22446E+00 SEY=0.1011E+OO COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.139E+01 TEST FOR SLOPES F, 1/ 10.= 0.380E+O0 TEST FOR LEVELS F, 1/ 11.= 0.268E+01 OVERALL TEST F, 2./ 10.= 0.145E+01 CORRELATION COEFFICIENT DEGREES OF FREEDOM R= 0.9822 R= 0.9852 R= 0.9789 DF= 6. DF= 6. DF= 13. TWO PERCENT MALT LINE 1 : NOTH LINE 2 : CHAE DAY DAY STANDARD ERROR INDIVIDUAL SIMREG LINES OF ESTIMATE 1 A= 0.12143E+00 B= 0.23250E+0O SEY=0.1052E+00 2 A= 0.36429E+00 B= 0.10714E+0O SEY=0.4629E-01 SIMREG FOR POOLED DATA 3 A = 0.24286E+00 B= 0.16982E+00 SEY=0.2082E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.516E+01 TEST FOR SLOPES F, 1/ 10.= 0.333E+02 TEST FOR LEVELS F, 1/ 11.= 0.900E+01 OVERALL TEST F, 2./ 10.= 0.344E+02 CORRELATION COEFFICIENT DEGREES OF FREEDOM R= 0.9822 DF= 6. R= 0.9837 DF= 6. 0.8697 DF= 13. TWO PERCENT MALT LINE 1 BAC DAY LINE 2 : B+C DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE CORRELATION COEFFICIENT DEGREES OF FREEDOM 1 A= 0.22286E+00 2 A= 0.26857E+00 B= 0.24536E+CO B= 0.21643E+00 SEY=0.1008E+00 SEY=0.8924E-01 SIMREG FOR POOLED DATA A= 0.24571E+OO B = O.23089E+0O SEY=0.9977E-01 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.128E+01 TEST FOR SLOPES F, 1/ 10.= 0.129E+01 TEST FOR LEVELS F, 1/ 11.= 0.184E+01 OVERALL TEST F, 2./ 10.= 0.159E+01 R= 0.9853 R= 0.9852 R= 0.9806 DF= 6. DF= 6. DF= 13. TWO PERCENT MALT LINE 1 : BAC LINE 2 DAY CHAE DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE CORRELATION COEFFICIENT DEGREES OF FREEDOM 1 A= 0.2228GE+00 2 A= O.36429E+0O B= 0.24536E+00 B= 0.10714E+0O SEY=0.1008E+00 SEY=0.4629E-01 SIMREG FOR POOLED DATA A= 0.29357E+00 B= 0.17625E+O0 SEY=0.2771E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.474E+01 TEST FOR SLOPES F, 1/ 10.= 0.435E+02 TEST FOR LEVELS F, 1/ 11.= O.198E+02 OVERALL TEST F, 2./ 10.= 0.699E+02 R= 0.9853 DF= 6. R= 0.9837 DF= 6. R= 0.8085 DF= 13. o TWO PERCENT MALT LINE 1 B+C DAY LINE 2 CHAE DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE A= 0.26857E+O0 A= 0.36429E+00 B= 0.21643E+O0 B= 0.10714E+00 SEY=0.8924E-01 SEY=0.4629E-01 SIMREG FOR POOLED DATA A = 0.31643E+00 B= O.16179E+00 SEY=0.2284E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.372E+01 TEST FOR SLOPES F, 1/ 10.= 0.331E+02 TEST FOR LEVELS F, 1/ 11.= 0.206E+02 OVERALL TEST F, 2./ 10.= 0.569E+02 CORRELATION COEFFICIENT DEGREES OF FREEDOM R= 0.9852 R= 0.9837 R= 0.8371 DF= 6. DF= 6. DF= 13. LIQUID COMPOST LINE 1 : NOTH LINE 2 : BAC DAY DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE CORRELATION COEFFICIENT DEGREES OF FREEDOM A= 0.45429E+00 A= 0.23143E+00 B= 0.16679E+00 B= 0.23G07E+00 SIMREG FOR POOLED DATA A= 0.34286E+00 B= 0.20143E+00 SEY=0.9208E-01 SEY=0.7203E-01 SEY=0.1102E+00 COVARIANCE ANALYSIS TESt FOR HOMOGENEITY OF RESIDUAL VARIANCES F. 5./ 5.= 0.163E+01 TEST FOR SLOPES F, 1/ 10.= 0.983E+01 TEST FOR LEVELS F, 1/ 11.= 0.837E+00 OVERALL TEST F. 2./ 10.= 0.5G7E+01 R= 0.9738 R= 0.9918 R= 0.9694 DF= 6. DF= 6. DF= 13. ro LIQUID COMPOST LINE 1 NOTH DAY LINE 2 B+F DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE CORRELATION COEFFICIENT DEGREES OF FREEDOM A= 0.45429E+00 A= 0.12286E+00 B= O.16679E+00 B= 0.24679E+00 SEY=0.9208E-01 SEY=O.S545E-01 SIMREG FOR POOLED DATA A = 0.28857E+00 B= 0.20679E+00 SEY=0.1187E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= O.116E+01 TEST FOR SLOPES F. 1/ 10.= O.114E+02 TEST FOR LEVELS F, 1/ 11.= 0.299E-01 OVERALL TEST F, 2./ 10.= 0.571E+01 R= 0.9738 R= 0.9895 R= 0.9665 DF= 6. DF= 6. DF= 13. co LIQUID COMPOST LINE 1 : NOTH LINE 2 : B+C DAY DAY STANDARD ERROR INDIVIDUAL SIMREG LINES OF ESTIMATE 1 A= 0.45429E+00 B= O.16G79E+00 SEY=0.9208E-01 2 A= O.35857E+0O B = O.15929E+0O SEY=0.4890E-01 SIMREG FOR POOLED DATA 3 A= 0.40643E+00 B= 0.16304E+OO SEY=0.9594E-01 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.355E+01 TEST FOR SLOPES F, 1/ 10.= O.145E+00 TEST FOR LEVELS F, 1/ 11.= 0.110E+02 OVERALL TEST F, 2./ 10.= 0.516E+01 CORRELATION COEFFICIENT DEGREES OF FREEDOM R= 0.9738 R= 0.9917 R = 0.9648 DF= 6. DF= 6. DF= 13. LIQUID COMPOST LINE 1 : NOTH LINE 2 : CHAE DAY DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE CORRELATION COEFFICIENT DEGREES OF FREEDOM A= 0.45429E+00 A= 0.57286E+00 B= 0.16679E+00 B= 0.48214E-01 SIMREG FOR POOLED DATA A= 0.51357E+00 B= 0. 10750E+00 SEY=0.9208E-01 SEY=0.2401E-01 SEY=0.2389E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F. 5./ 5.= 0.147E+02 TEST FOR SLOPES F, 1/ 10.= 0.435E+02 TEST FOR LEVELS F. 1/ 11.= 0.201E+02 OVERALL TEST F, 2./ 10.= 0.706E+02 R= 0.9738 DF= 6. R= 0.9786 DF= 6. R= 0,6970 DF= 13. LIQUID COMPOST LINE 1 BAC DAY LINE 2 : B+F DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE 1 A= 0.23143E+00 2 A= O.12286E+00 B= 0.23607E+00 B= 0.24679E+00 SEY=O.7203E-O1 SEY=0.8545E-01 SIMREG FOR POOLED DATA A = 0.17714E+00 B= 0.24143E+0O SEY=0.8123E-01 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.710E+00 TEST FOR SLOPES F, 1/ 10.= 0.257E+00 TEST FOR LEVELS F, 1/ 11.= 0.260E+01 OVERALL TEST F. 2./ 10.= 0.134E+01 CORRELATION DEGREES OF COEFFICIENT FREEDOM R= 0.9918 DF= 6. R= 0.9895 DF= 6. R= 0.9881 DF= 13. LIQUID COMPOST LINE 1 BAC DAY LINE 2 B+C DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE A= 0.23143E+00 A= O.35857E+0O B= 0.23607E+00 B= O.15929E+00 SEY=0.7203E-01 SEY=0.4890E-O1 SIMREG FOR POOLED DATA A= O.295O0E+0O B= 0.19768E+00 SEY=0.1396E+CO COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.217E+01 TEST FOR SLOPES F, 1/ 10.= 0.218E+02 TEST FOR LEVELS F, 1/ 11.= 0.104E+02 OVERALL TEST F, 2./ 10.= 0.259E+02 CORRELATION DEGREES COEFFICIENT FREEDOM R= 0.9918 DF= 6. R= 0.9917 DF= 6. R= 0.9505 DF= 13. LIQUID COMPOST LINE 1 : BAC LINE 2 : CHAE DAY DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE CORRELATION COEFFICIENT DEGREES OF FREEDOM A= 0.23143E+00 A= 0.57286E+00 B= 0.23607E+00 B= 0.48214E-01 SEY=0.7203E-01 SEY=0.2401E-01 SIMREG FOR POOLED DATA A= 0.40214E+00 B= 0.14214E+00 SEY=0.3043E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.900E+01 TEST FOR SLOPES F, 1/ 10.= O.171E+03 TEST FOR LEVELS F, 1/ 11.= O.124E+02 OVERALL TEST F. 1.1 10.= 0.188E+03 R= 0.9918 R= 0.9786 R= 0.7103 DF= 6. DF= 6. DF* 13. o v CO LIQUID COMPOST LINE 1 : B+F DAY LINE 2 B+C DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE CORRELATION COEFFICIENT DEGREES OF FREEDOM A= O.12286E+00 A= 0.35857E+00 B = 0.24679E+00 B= 0.15929E+00 SEY=0.8545E-01 SEY=0.4890E-01 SIMREG FOR POOLED DATA A= 0.24071E+OO B= 0.20304E+00 SEY=0.1295E+O0 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.305E+01 TEST FOR SLOPES F, 1/ 10.= 0.221E+02 TEST FOR LEVELS F, 1/ 11.= 0.323E+01 OVERALL TEST F. 2./ 10.= 0.158E+02 R= 0.9895 R= 0.9917 R= 0.9590 DF= 6. DF= 6. DF= 13. (£> LIQUID COMPOST LINE 1 B+F DAY LINE 2 : CHAE DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE 1 A= 0.12286E+00 2 A= 0.57286E+00 B= 0.24679E+00 B= 0.48214E-01 SIMREG FOR POOLED DATA A= 0.34786E+00 B= 0.14750E+00 SEY=0.8545E-01 SEY=0.2401E-01 SEY=0.2896E+O0 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F, 5./ 5.= 0.127E+02 TEST FOR SLOPES F, 1/ 10.= O.140E+03 TEST FOR LEVELS F, 1/ 11.= 0.772E+01 OVERALL TEST F. 2./ 10.= O.123E+03 CORRELATION COEFFICIENT DEGREES OF FREEDOM R= 0.9895 R= 0.9786 R= 0.7400 DF= 6. DF= 6. DF= 13. LIQUID COMPOST LINE 1 : B+C LINE 2 : CHAE DAY DAY INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE 1 A= 0.35857E+00 2 A= 0.57286E+00 B= 0.15929E+00 B= 0.48214E-01 SEY=0.4890E-01 SEY=0.2401E-01 SIMREG FOR POOLED DATA A= 0.46571E+00 B= 0.1O375E+O0 SEY=0.1762E+CO COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F. 5./ 5.= 0.415E+01 TEST FOR SLOPES F, 1/ 10.= 0.116E+03 TEST FOR LEVELS F, 1/ 11.= 0.109E+02 OVERALL TEST F, 2./ 10.= O.121E+03 CORRELATION DEGREES OF COEFFICIENT FREEDOM R= 0.9917 DF= 6. R= 0.9786 DF= 6. R= 0.7861 DF= 13. APPENDIX B Statistical analysis of mushroom yields. VARIABLE NAMES - YIELD DATA FORMAT (I2.4X.F6.1) YIELD CONVENTIONAL SOURCE TREAT ERROR TOTAL DF 3 7 10 ANALYSIS OF VARIANCE - YIELD SUM SQ MEAN SQ 14500. 2763.4 17264. 4833.4 394.77 ERROR F-VALUE 12.244 PROB 0.35742E-02 GRAND MEAN 144.60 STANDARD DEVIATION OF VARIABLE 1 IS 41.550 FREQUENCIES. MEANS. STANDARD DEVIATIONS ******************************************************************************** TREAT 1 2 3 4 MN YIELD 172.2 170.8 138.0 73.80 STUDENTIZED RANGES FOR NEWMAN-KEUL'S TEST, ALPHA=0.05 3.344 4.165 4.681 THERE ARE 2 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS. NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE FOR A SUBSET OF THAT SIZE) WHICH ARE LISTED AS FOLLOWS ( 4 ) ( 3, 2, 1) TIME FOR MULTIPLE RANGE TESTS IS 0.1180E-01' SECONDS. ANALYSIS COMPLETE. OJ VARIABLE NAMES - YIELD DATA FORMAT (I2.4X.F6.1) YIELD - TWO PERCENT MALT SOURCE TREAT ERROR TOTAL ANALYSIS OF VARIANCE - YIELD DF SUM SO MEAN SO ERROR F-VALUE 3 8 11 27. 189 15.893 43.082 9.0631 1 .9867 4.5619 PROB 0.38232E-01 GRAND MEAN 2.1250 STANDARD DEVIATION OF VARIABLE 1 IS 1.9790 FREQUENCIES, MEANS, STANDARD DEVIATIONS ***************************************************************** TREAT MN YIELD 1 .500 3.067 3 4 3.933 0.0 STUDENTIZED RANGES FOR NEWMAN-KEUL'S TEST. ALPHA=0.05 3.261 4.041 4.529 THERE ARE 2 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS, NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE FOR A SUBSET OF THAT SIZE) WHICH ARE LISTED AS FOLLOWS ( 4. 1, 2) ( 1. 2, 3) TIME FOR MULTIPLE RANGE TESTS IS 0.8255E-02 SECONDS. ANALYSIS COMPLETE. -•4 VARIABLE NAMES - YIELD DATA FORMAT (I2.4X.F6.1) YIELD - LIQUID COMPOST SOURCE TREAT ERROR TOTAL DF 4 10 14 ANALYSIS OF VARIANCE - YIELD SUM SQ MEAN SQ 444.98 47.587 492.57 111.25 4.7587 ERROR F-VALUE 23.377 PROB 0.46429E-04 GRAND MEAN 9.6733 STANDARD DEVIATION OF VARIABLE 1 IS 5.9316 FREQUENCIES. MEANS, STANDARD DEVIATIONS ******************************************************************************** TREAT 1 2 3 4 5 MN YIELD 9.000 12.30 10.57 16.50 0.0 STUDENTIZED RANGES FOR NEWMAN-KEUL'S TEST, ALPHA=0.05 3. 151 3.877 4.327 4.654 THERE ARE 3 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS, NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE FOR A SUBSET OF THAT SIZE) WHICH ARE LISTED AS FOLLOWS ( 5) ( 1. 3. 2) ( 4) TIME FOR MULTIPLE RANGE TESTS IS 0.8568E-02 SECONDS. ANALYSIS COMPLETE. -~4 tn 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0095833/manifest

Comment

Related Items