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

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BIOLOGICAL C O N T R O L OF OLIVE CREEN  MOLD  IN T H E CULTIVATION  OF AGARICUS  BISPORVS  by THOMAS EDWARD B.Sc,  TAUTORUS  University of British Columbia, 1979  A THESIS SUBMITTED  IN P A R T I A L FULFILLMENT OF  T H E REQUIREMENTS  FOR T H E DEGREE OF  MASTER O F S C I E N C E in T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Microbiology  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH  COLUMBIA  April 1983 (c)  Thomas Edward Tautorus,  1983  In p r e s e n t i n g  this  thesis i n partial  fulfilment of the  r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y of B r i t i s h Columbia, I agree that it  freely  t h e L i b r a r y s h a l l make  a v a i l a b l e f o r r e f e r e n c e and study.  agree that permission f o r extensive for  copying of t h i s  understood that financial  copying o r p u b l i c a t i o n o f t h i s  gain  Department  of  MICROBIOLOGY  The U n i v e r s i t y o f B r i t i s h 1956 Main M a l l V a n c o u v e r , Canada V6T 1Y3  DE-6 n / a n  It i s thesis  s h a l l n o t be a l l o w e d w i t h o u t my  permission.  Date  thesis  s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d 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 . for  I further  A p r i l 20 / 1983  Columbia  written  ABSTRACT  T h e C a n a d i a n mushroom i n d u s t r y i s e x p e r i e n c i n g t h e same p r o b l e m s as a r e e n c o u n t e r e d w o r l d w i d e - a t t a c k s o f d i s e a s e s a n d p e s t s ( l n g r a t t a , 1980).  S u c c e s s f u l methods to c o n t r o l t h e d a m a g i n g weed mold -  Chaetomium olivaceum ( O l i v e g r e e n mold) i n mushroom b e d s a r e p r e s e n t l y not k n o w n .  T h i s t h e s i s i n v e s t i g a t i o n a t t e m p t e d to c o n t r o l C. olivaceum  b y b i o l o g i c a l means.  A t h e r m o p h i l i c B a c i l l u s s p ( r e s e m b l i n g B. coagu-  lans - r e s i s t a n t to 0.02% sodium a z i d e , a c i d o p h i l i c ) w h i c h s h o w e d d r a m a t i c a c t i v i t y a g a i n s t C. olivaceum on T S Y  ( T r y p t i c a s e soy a g a r + 0.1% Y e a s t  e x t r a c t ) a g a r p l a t e s was i s o l a t e d from commercial mushroom compost (phase I ) .  When i n o c u l a t e d i n t o c o n v e n t i o n a l a n d h y d r o p o n i c  mushroom  b e d s , t h e B a c i l l u s not o n l y p r o v i d e d a s i g n i f i c a n t d e g r e e o f p r o t e c t i o n from C. olivaceum b u t also i n c r e a s e d y i e l d s o f Agaricus bisporus.  This  i s t h e f i r s t i s o l a t i o n o f a m i c r o o r g a n i s m i n h i b i t o r y to O l i v e g r e e n mold. T h e B a c i l l u s was s h o w n to p r o d u c e an e x t r e m e l y p o t e n t a n d s t a b l e a n t i b i o t i c (named C h a e t o m a c i n ) e f f e c t i v e o v e r a w i d e r a n g e o f b o t h p H (2-10) a n d t e m p e r a t u r e (-15°C to 150°C).  C h a e t o m a c i n i s s o l u b l e i n most  polar solvents and insoluble in non-polar solvents.  This antibiotic  p r o d u c e d at m e s o p h i l i c t e m p e r a t u r e s i s also a c t i v e a g a i n s t o t h e r B a c i l l u s s p e c i e s a n d v a r i o u s e u k a r y o t e s - b u t d e m o n s t r a t e s no a c t i v i t y a g a i n s t Cram negative organisms or Gram positive cocci.  Final purification of  C h a e t o m a c i n was a c c o m p l i s h e d t h r o u g h t h i n l a y e r c h r o m a t o g r a p h y  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 ii  ABSTRACT 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 5.  20  Fruiting  ....  24  B.  Weed Molds (Chaetomium olivaceum)  26  C.  Biological Control  32  METHODS AND MATERIALS I. II.  37  Selection of Thermophiles  37  Identification  42  of Thermophiles  1.  Microscopic Appearance  42  2.  Macroscopic Appearance  42  3.  Motility  « iv  Page 4. Anaerobic Growth  43  5. Maximum and Minimum Temperatures 6. III.  IV.  of Growth .  Biochemical Reactions  44 44  Use of Bacillus AOG in Mushroom Cultivation  48  A.  Conventional Production of Agaricus bisporus ..  48  B.  Hydroponic Production of Agaricus bisporus  50  A n a l y s i s of Inhibitor Produced by Bacillus AOG A.  Determination of a pH Change  B.  Methods of Extracting free Extracts i) ii) iii)  ...  54 54  Inhibitor from Cell-  of Bacillus AOG  55  thermophilic conditions  55  mesophilic conditions  57  evaporation  58  method  59  C.  Temperature  Stability  D.  pH Stability  59  E.  Solvent Solubility  60  F.  Spectrum of Activity  G.  Thin-layer  H.  Ultraviolet Spectrum  65  I.  Column Chromatography  66  Chromatography  i) Ion-exchange ii)  of Antibiotic  61 61  66  Slephadex LH 20  67  J.  Fluorescent Spectrum  68  K.  Amino Acid Analysis  68  v.  Page RESULTS I.  II. III.  AND DISCUSSION  69  Isolation of Thermophiles Antagonistic  Towards  Chaetomium olivaceum  69  Identification  73  Cultivation  of Isolated Thermophiles  of Agaricus bisporus  with the  Thermophile  Bacillus AOG  IV.  83  1.  Conventional Methods  83  2.  Hydroponic Methods  86  Analysis of Inhibitor Produced by Bacillus AOG  97  A.  Effect of pH  97  B.  Extraction of Antibiotic  98  i) ii)  Thermophilic extraction  98  Mesophilic extraction  99  C.  Temperature  D.  Solvent Solubility  103  E.  Spectrum of Activity  105  F.  Thin-layer  105  G.  Column Chromatography i)  and pH Stability of Antibiotic  Chromatography  Ion-exchange  ii) Sephadex  LH 20  103  110 110 110  H.  Ultraviolet Spectrum  113  I.  Fluorescent  113  J.  Amino Acid Analysis  Spectrum  vi.  116  Page CONCLUSIONS  123  LITERA T URE CITED  126  APPENDICES  142  A.  Statistical Analysis of Mycelial Diameters  143  Statistical  172  B.  ;  Analysis of Mushroom Yields  vii.  LIST OF TABLES  Table  Page  1.  Microorganisms  tested, for susceptibility  2.  Microscopic observations  to antibiotic  62  and colony morphology of  isolated thermophiles  74  3.  Properties  76  4.  Summary of properties  of isolated thermophiles of known thermophilic  Bacillus  species  78  5.  Extraction  6.  Solvent solubility  7.  Solvent systems investigated  8.  of inhibitor from TSY agar discs of antibiotic  100 104  to determine optimum TLC  separation of crude antibiotic  107  Amino acid analysis of Bands I and II  117  viii.  LIST OF FIGURES  Figure 1.  P  Extensive  Selection of thermophiles for activity  against 41  Aerobic waste fermenter used for preparation of liquid compost  4.  51  Ultraviolet spectra of lignin extracted from wheatstraw and compost  5.  70  Thermophiles showing varying  degrees of antagonism  towards C_. olivaceum on TSY agar 6.  Gram reaction of Thermophile #10  7.  Inhibition of Olive green mold on TSY agar by  72 75  Bacillus AOG  82  8.  Mycelial development  9.  Yield of mushrooms in standard  in standard  mushroom compost  Mycelial development  11.  Poor spawn growth in hydroponic  85  in 2% Malt extract  Improved mycelial development  ,  89  due to the biological  of Bacillus AOG against C_. olivaceum  ix.  88  tray with only  Olive green mold present  protection  84  mushroom compost  10.  12.  e  30  Chaetomium olivaceum 3.  g  contamination of mushroom compost bed  by Chaetomium olivaceum 2.  a  89  Figure 1 3.  Pq.ge Yield of mushrooms in 2% Malt extract  90  14.  Mycelial development  15.  Yield of mushrooms in liquid compost  16.  Fermentation of 2% Malt extract by Bacillus AOG  17.  Inhibition of C_. olivaceum from cell-free  18.  in liquid compost  91 92 94  extracts of  Bacillus AOG  ,  Spectrum of activty of Bacillus AOG against  various  102  microorganisms  106  19.  Antibiotic  111  20.  Ultraviolet absorption  21.  elution profile in cation exchange resin spectra of lower (Band I) and  upper (Band II) TLC bands  114  Fluorescent spectrum of TLC Band I  115  x.  ACKNOWLEDGEMENTS  The author wishes to express his sincere appreciation Townsley,  Dept. of Food Science,  and enthusiasm throughout  to Dr. P.M.  for his invaluable advice,  guidance  the course of this study.  He is also thankful to the members of his committee, Dr. Campbell, Dr. A. Ells, and Dr. D. Syeklocha, patience,  for their interest  in,  and review of this thesis.  Thanks are also extended to Mr. S. Yee for'his assistance  J.J.R.  and Mr. R. Yada for statistical  The author is extremely grateful encouragement,  inspiring  criticism,  technical  analysis.  to Ms. Cheryl Craig for her  and her endless work in  preparing  the artwork and manuscript of this thesis. The support acknowledged.  of the B. C. Dept. of Agriculture  is also gratefully  1. INTRODUCTION  The  c u l t i v a t i o n o f t h e common w h i t e m u s h r o o m , Agaricus  L a n g e {Agaricus brunnescens) years.  has e x p a n d e d t r e m e n d o u s l y  bisporus  i n recent  T h e B r i t i s h C o l u m b i a M i n i s t r y o f A g r i c u l t u r e (1981) s t a t e s  t h a t 8820 t o n s o f A. bisporus  were produced within this province  i n 1981 ( r e p r e s e n t i n g a p p r o x i m a t e l y  a 300 t o n i n c r e a s e o v e r  M u s h r o o m s c o n t i n u e t o r a n k s e c o n d i n economic i m p o r t a n c e v e g e t a b l e c r o p i n B.C.  (third overall in Canada).  1980). as a  Furthermore,  B r i t i s h C o l u m b i a n s lead t h e c o n t i n e n t ( a n d p o s s i b l y t h e w o r l d ) i n mushroom c o n s u m p t i o n p e r c a p i t a , c o n s u m i n g t w i c e t h a t o f fellow C a n a d i a n s a n d t h r e e times as m u c h a s U.S. c o n s u m e r s ( A n o n . , 1981; P o t t e r , 1980).  It i s l i k e l y t h a t t h e mushroom i n d u s t r y w i l l become  of g r e a t e r importance  i n the production of enzymes, antitumor  c o m p o u n d s , a n d a n t i b i o t i c s , a s well as food a n d feed ( K u r t z m a n , 1979). H o w e v e r , i r r e s p e c t i v e o f p u b l i c d e m a n d a n d food v a l u e s , t h e f u t u r e prospects f o r continued expansion  a n d mass p r o d u c t i o n l a r g e l y  concerns  t h e economics o f p r o d u c t i o n methods ( H a y e s S N a i r , 1975). T r a d i t i o n a l l y a c c e p t e d as a h o r t i c u l t u r a l c r o p , mushroom production is in principle a fermentation process K u r t z m a n , 1979).  ( H a y e s , 1974;  P r e s e n t l y mushroom c u l t u r e r e p r e s e n t s t h e o n l y  major p r o c e s s i n b i o t e c h n o l o g y w h i c h s u c c e s s f u l l y c o n v e r t s c e l l u l o s i c s i n t o u s e f u l foods a n d b y p r o d u c t s . procedures:  One cycle consists of distinct  namely s u b s t r a t e p r e p a r a t i o n , i n o c u l a t i o n , i n c u b a t i o n .  2.  g r o w t h , a n d t e r m i n a l d i s i n f e c t i o n ; all o f w h i c h c a n b e i d e n t i f i e d w i t h s t a n d a r d l a b o r a t o r y methodology employed 1979).  i n m i c r o b i o l o g y a n d i n t h e methods  i n t h e f e r m e n t a t i o n i n d u s t r y ( H a y e s , 1974; H a y e s & W r i g h t , T h e p r i n c i p l e s i n v o l v e d i n c u l t i v a t i o n a r e common t o o t h e r  industrial processes involving microorganisms, the guarantee o f purity of culture which in turn, provides for the guarantee o f e d i b i l i t y - is t h e basis on which t h e i n d u s t r y was founded. The changed  methods used i n this solid-state significantly over the years.  f e r m e n t a t i o n h a v e not  T h e main s u b s t r a t e w h i c h  s u p p o r t s mushroom g r o w t h c o n t i n u e s to c o n s i s t o f animal m a n u r e s , plant materials, chemical f e r t i l i z e r s , a n d other a g r i c u l t u r a l  residues.  It must b e k e p t i n mind t h a t mushroom compost i s 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 distinguished b y differences in physical requirements, productsof metabolism, a n d v a r i o u s n u t r i t i o n a l r e q u i r e m e n t s ( L a m b e r t , 1938).  More-  o v e r , with the h i g h labor input and the p u r e 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 little to offset c r o p e r r o r and failure due to  diseases and pests. F u n g i w h i c h g r o w i n c o m p e t i t i o n w i t h t h e mushroom mycelium a r e r e f e r r e d to as weed molds a n d a r e c o n s i d e r e d s e r i o u s d i s e a s e c a u s i n g a g e n t s ( L a m b e r t S A y e r s , 1 953).  O n e s u c h o r g a n i s m i s Chaetomium  olivaceum more commonly k n o w n as O l i v e g r e e n mold.  Olive green  is a weed mold w h i c h f r e q u e n t l y o c c u r s a f t e r p a s t e u r i z a t i o n o f t h e compost a n d s e v e r e l y i n h i b i t s mushroom m y c e l i a l d e v e l o p m e n t b y c o m p e t i n g f o r n u t r i e n t s ( B e a c h , 1937).  C o n s e q u e n t l y , mushroom  mold  3.  y i e l d s may b e completely d e s t r o y e d .  O n e reason f o r the invasion of  C. olivaceum into mushroom b e d s is t h e p r e s e n c e o f f r e e ammonia i n t h e compost a f t e r t h e p h a s e II p a s t e u r i z a t i o n ( R e t t e w , o x y g e n d u r i n g p h a s e II  1948).  Insufficient  leads to a n a e r o b i c decomposition o f t h e compost.  T h i s p r o d u c e s c o m p o u n d s t o x i c to mushrooms while e n c o u r a g i n g colonization o f t h e compost b y O l i v e g r e e n mold ( N a i r ,  1980).  y e t , t h e r e a r e no k n o w n methods to s u c c e s s f u l l y c o n t r o l t h i s (Vedder,  A s of pest.  1978).  Economic y i e l d s r e q u i r e that a g r o w e r u s e t h e b e s t t e c h n i q u e s p e r t a i n i n g to f u n g a l p h y s i o l o g y a n d d i s e a s e p r o t e c t i o n . research (Huhnke, 1970; T o w n s l e y ,  1970; Nair & F a h y , 1972; S t a n e k  From e a r l i e r  & Rysava-Zatecka,  1974), it was s u g g e s t e d that a c e r t a i n d e g r e e o f  p r o t e c t i o n from i n v a s i o n o f t h e mushroom compost b y d i s e a s e  causing  o r g a n i s m s may b e o b t a i n e d b y p r i o r f e r m e n t a t i o n with s e l e c t e d m i c r o organisms.  H u h n k e (1970)  philic microorganisms  states t h a t , b y i n o c u l a t i n g s p e c i f i c t h e r m o -  into s t e r i l i z e d compost t h e c u l t u r e s a r e c a p a b l e  of c a u s i n g a s e l e c t i v e p r o t e c t i o n o f t h e s u b s t r a t e a g a i n s t d i s e a s e s a n d pests.  H e n c e t h e objectives o f t h i s t h e s i s i n v e s t i g a t i o n were to  select s p e c i f i c t h e r m o p h i l i c m i c r o o r g a n i s m s w h i c h would not o n l y  support  the m u s h r o o m , b u t p r o t e c t it from damage from O l i v e g r e e n mold. F u r t h e r m o r e , it was h o p e d that t h i s t h e r m o p h i l e ( s )  could be used in the  p r e p a r a t i o n o f a h y d r o p o n i c system u t i l i z i n g s y n t h e t i c s u b s t r a t e s b y e l i m i n a t i n g t h e potential h a z a r d s o f compost) Agaricus  bisporus.  (there-  as a g r o w t h medium f o r  If s u c c e s s f u l , t h e r e s u l t s o f t h i s r e s e a r c h s h o u l d  facilitate t h e e m e r g e n c e o f t h e mushroom i n d u s t r y into a level o f t e c h n o l o g y e x p e r i e n c e d i n modern i n d u s t r i a l f e r m e n t a t i o n s .  4.  L I T E R A T U R E REVIEW  A . C U R R E N T 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).  T h e 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, p i g , 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).  T h e 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 microorganisms 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 temperatures 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, polysaccharides, 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.  s u c h as c e l l u l o s e a n d l i g n i n .  T h e compost p i l e s a r e t u r n e d f o u r times  o v e r a p e r i o d o f 8-9d to m a i n t a i n a e r o b i c c o n d i t i o n s w i t h i n t h e s t a c k . T h e d u r a t i o n of time s p e n t on p h a s e I w i l l d e p e n d on many local f a c t o r s air temperature, t y p e of composting s u b s t r a t e s , moisture, and microflora present. T h e l i t e r a t u r e d e s c r i b i n g t h e p r o g r e s s i v e c h e m i c a l c h a n g e s accompa n y i n g d e c o m p o s i t i o n o f o r g a n i c m a n u r e s has been e x t e n s i v e l y r e v i e w e d ( 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; K u r t z m a n , M a e d e r , 1960; M a t t i n g l y , 1952; M u l l e r , 1965; S c h i s l e r , 1980;  1979;  Schobinger,  1958; Waksman & C o r d o n , 1939; Waksman et al,1939; Waksman & I y e r , 1932a,b; Waksman & M c G r a t h ,  1931; Waksman & N i s s e n , 1931,  1932).  C e l l u l o s e , h e m i c e l l u l o s e , a n d s o l u b l e s all d e c r e a s e w h i l e l i g n i n i n c r e a s e s o v e r the course of composting. are  Since both lignin and insoluble proteins  r e a d i l y a v a i l a b l e to mushroom mycelium  (Waksman & N i s s e n , 1932b)  a n d not so r e a d i l y u s e a b l e to r i v a l m i c r o o r g a n i s m s , i t seems p r o b a b l e t h a t t h e b u i l d u p o f t h e s e p r o d u c t s at t h e e x p e n s e o f t h e more e a s i l y d e c o m p o s e d c a r b o h y d r a t e s a n d s o l u b l e p r o t e i n s i s an i m p o r t a n t component in t h e c o m p l e x p h e n o m e n a r e s p o n s i b l e f o r t h e f i n i s h e d compost a t t a i n i n g a balance advantageous  f o r t h e mushroom  mycelium.  T h e o b j e c t i v e s o f t h e mushroom g r o w e r i s to r e g u l a t e c h e m i c a l a n d p h y s i c a l c o n d i t i o n s d u r i n g f e r m e n t a t i o n so t h a t t h e f i n i s h e d compost w i l l be s u i t a b l e f o r mushroom mycelium to p r e d o m i n a t e o v e r all o f t h e c o m p e t i t i v e m i c r o b i a l f l o r a p r e s e n t ( F o r d y c e , 1970; L a m b e r t , R o y s e & S c h i s l e r , 1980; S c h i s l e r , 1980).  1938;  The c a r b o h y d r a t e s that are  e a s i l y b r o k e n d o w n must be r e m o v e d so t h a t o t h e r f u n g i w i l l be less apt  7.  to g r o w i n c o m p e t i t i o n ( V e d d e r , 1978).  H a y e s (1969) a n d F e r m o r et a l  (1979) s t a t e t h a t t h i s c a n be a c c o m p l i s h e d b y e n c o u r a g i n g t h e g r o w t h o f l a r g e t h e r m o p h i l i c m i c r o b i a l p o p u l a t i o n s to u t i l i z e t h e s i m p l e c a r b o h y d r a t e s w h i c h w o u l d o t h e r w i s e be a v a i l a b l e to be u s e d b y u n d e s i r a b l e m e s o p h i l i c f u n g i . ' B e c a u s e i o f t h e lower m e s o p h i l i c n u m b e r s , less c e l l u l o s e  a n d h e m i c e l l u l o s e a r e u t i l i z e d b y them; c o n s e q u e n t l y  t h e r e is a n e t c o n s e r v a t i o n o f n u t r i e n t s w h i c h s u p p o r t mushroom g r o w t h . T h e u n d e r s t a n d i n g o f t h e components which f u n c t i o n i n composting led to e x p e r i m e n t s d e s i g n e d to s h i f t t h e p a t t e r n o f t h e m i c r o b i o l o g i c a l sequence i n the composting fermentation i n favor of the organisms which a r e u n a b l e to b r e a k d o w n c e l l u l o s e a n d h e m i c e l l u l o s e .  I n c r e a s e d mushroom  y i e l d s w e r e o b t a i n e d from s e v e r a l w o r k e r s ( H a y e s , 1969; H a y e s & R a n d l e , 1969a,b; L a b o r d e et a l , 1972; S m i t h , 1974) b y t h e a d d i t i o n o f a c a r b o h y d r a t e s u p p l e m e n t at t h e b e g i n n i n g o f p h a s e I. T h e a d d i t i o n o f n u t r i e n t s at p h a s e I i s d i r e c t e d t o w a r d s t h e f e e d i n g o f m i c r o b i a l p o p u l a t i o n s i n t h e compost r a t h e r t h a n d i r e c t n u t r i e n t a d d i t i o n f o r t h e m u s h room.  S u c r o s e s u p p l e m e n t a t i o n ( H a y e s , 1969, 1977) f a v o r e d h i g h  p o p u l a t i o n s o f b a c t e r i a , at t h e e x p e n s e o f a c t i n o m y c e t e s , w i t h 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 conserv a t i o n o f n u t r i e n t s w h i c h was a s s o c i a t e d w i t h i n c r e a s e d y i e l d .  Furthermore,  s u p p l e m e n t a t i o n p r o b a b l y c a u s e d a n i n c r e a s e i n b a c t e r i a l biomass ( i e . e x t r a c e l l u l a r p o l y s a c c h a r i d e c a p s u l e s ) w h i c h i s a major s o u r c e o f n u t r i e n t s f o r A.  bisporus  ( E d d y & J a c o b s , 1976; S t a n e k , 1972).  S a n A n t o n i o (1966)  c o n c l u d e d t h a t m i c r o o r g a n i s m s may a c c o u n t f o r e r r a t i c r e s u l t s o b t a i n e d , w h e n d i f f e r e n t amounts o f s u p p l e m e n t s w e r e a d d e d .  8.  B a c t e r i a a r e p r o b a b l y more important to the mushroom t h a n to p r o d u c e r s o f a n y o t h e r c r o p ( F l e t c h e r , 1979).  grower  T h e y play a  s i g n i f i c a n t role d u r i n g the p r e p a r a t i o n o f t h e c o m p o s t , initiation o f f r u i t b o d i e s , a n d may b e a major problem b e c a u s e o f t h e d i s e a s e s produce.  A l l major g r o u p s o f microorganisms  are active d u r i n g  they composting,  e s p e c i a l l y t h e r m o p h i l i c b a c t e r i a , f u n g i , a n d a c t i n o m y c e t e s , a n d ideally each g r o u p is dominant at d i f f e r e n t s t a g e s . Waksman S C o r d o n (1939) a n d Waksman et al (1939b,c)  were the f i r s t  to emphasize the i m p o r t a n c e in mushroom c o m p o s t i n g o f a mixed m i c r o f l o r a of t h e r m o p h i l i c a c t i n o m y c e t e s , b a c t e r i a , a n d f u n g i .  Since t h e n , there have  been a g r e a t deal o f s t u d i e s c o n c e r n i n g t h e role o f m i c r o o r g a n i s m s p r e p a r a t i o n o f compost ( H a y e s , 1969, 1977; O l i v e r S G u i l l a u m e s , Stanek,  Stanek,  1976;  1972) a n d o f the p r e d o m i n a n t t y p e s a n d s u c c e s s i o n o f each ( C h a n g  & Hudson, Hayes,  in the  1967; F e r g u s ,  1964; F e r m o r et a l , 1979; F o r d y c e , 1970;  1969, 1977; L a b o r d e et a l , 1968; R e n o u x - B l o n d e a u , 1967, 1972; S t a n e k S Z a t e c k a ,  Hayes  1959;  1967).  (1969) examined the b a c t e r i a l p o p u l a t i o n s d u r i n g p h a s e I a n d  r e c o r d e d the s u c c e s s i o n a n d ecological c y c l e o f the major t y p e s . b e g i n n i n g o f c o m p o s t i n g Bacillus  subtilus  and a Flavobacterium p r e -  d o m i n a t e d , b u t a f t e r the t h i r d d a y B. stearothermophilus monas s p . became the major t y p e s .  A t the  and Pseudo-  T h e r m o p h i l i c f u n g i remain a c t i v e  t h r o u g h o u t the p r o c e s s a n d t h e y c o n d i t i o n the v e g e t a b l e matter f o r lateruutilization b y the mushroom ( e g . H u m i c o l a , M u c o r , S t i l b e l l a ) .  He  noted that all c o m p o s t s , within n a r r o w limits, a r e o f a g i v e n microbiological composition.  O l i v e r & Guillaumes  (1976) f o u n d that the o c c u r r e n c e o f 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 s p . , 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 humuslignin fractions (ie. lignin-N complex) (Muller, 1965; Schisler, 1980). remainder is probably fixed in microbial protein.  The  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 g r o w t h and a heat p r o d u c i n g f e r m e n t a t i o n .  Further-  more, t h e majority of l i p i d s u s e d b y t h e mushroom from t h e compost a p p e a r to be i n t r a c e l l u l a r l i p i d s o f t h e r m o p h i l i c m i c r o f l o r a s 1980). this.  T h e f a t t y a c i d composition of t h e r m o p h i l i c f u n g i  (Schisler,  substantiates  It has been s u g g e s t e d that p r o t e i n a n d acetate u n i t s a r e o b t a i n e d  b y t h e mushroom p r i m a r i l y from the biomass of t h e r m o p h i l e s b u i l t u p d u r i n g the p h a s e I s t a g e ( H a y e s ,  1974).  A n u m b e r o f t h e r m o p h i l e s h a v e been a d d e d to compost in e f f o r t s to i n c r e a s e the s p e e d o f p h a s e I o r to i n c r e a s e the y i e l d o f  mushrooms.  Pope et al (1962) a d d e d t h e r m o p h i l i c f u n g i d u r i n g t h e c o m p o s t i n g and Renoux-Blondeau groups  (1959) a d d e d actinomycetes a f t e r p h a s e II  r e p o r t e d i n c r e a s e s in y i e l d .  Stanek & Rysava-Zatecka  process - both  (1970)  a d d e d mixed c u l t u r e s of t h e r m o p h i l i c a c t i n o m y c e t e s a n d b a c t e r i a to s t e r i l i z e d compost.  T h e y f o u n d mushroom c u l t u r e s were not contaminated  by competitive microorganisms their presence.  a n d mushroom mycelium g r e w well in  F u r t h e r m o r e , H u h n k e (1970) states that b y i n o c -  ulating specific thermophilic microorganisms c u l t u r e s a r e c a p a b l e of c a u s i n g  into s t e r i l i z e d compost,  the  ( t h r o u g h p r o d u c t i o n of metabolic b y -  p r o d u c t s , enzymes, etc.) a selective protection of the substrate against diseases a n d  pests.  It would a p p e a r that t h e r e is a g r e a t e f f o r t to f i n d ways to r e d u c e the amount of time r e q u i r e d f o r c o m p o s t i n g 1979).  ( F e r m o r et a l , 1979; K u r t z m a n ,  H o w e v e r , most of the r a p i d c o m p o s t i n g methods at some time  h a v e had d i f f i c u l t i e s with d i s e a s e and weed f u n g i ( C h a n t e r & S p e n c e r , 1974).  It a p p e a r s that e i t h e r t h e y can not r e p r o d u c i b l y remove the easily  12.  metabolized nutrients or that there are some naturally occurring antibiotics produced during composting, which are not adequately produced in t h e 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  ( T u r n e r , 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). about 55-60°C.  Steam is initially added to bring the temperature to 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. fermentation (phase II)  The  must remove the last of the easily available  14.  n u t r i e n t s - p r o d u c i n g a compost w h i c h is a s e l e c t i v e medium f o r the mushroom ( E d d y S J a c o b s , 1976).  S c h i s l e r (1980) states that c o n v e r s i o n  o f ammonia p r o d u c t s to microbial p r o t e i n is b e s t a c c o m p l i s h e d b y thermophilic b a c t e r i a , actinomycetes (eg. Streptomyces, and fungi ( T o r u l a , Mucor).  Thermonopsora)  B y so d o i n g , t h e ammonia is i n c o r p o r a t e d into  microbial cells a n d ultimately is a v a i l a b l e to the mushroom ( S i n d e n S Hauser,  1953).  H o w e v e r , the p o s s i b l e p r o d u c t i o n of metabolites o r  compost d e g r a d a t i v e p r o d u c t s that i m p r o v e o r r e d u c e ultimate mushroom y i e l d is also t h e o r e t i c a l l y p o s s i b l e ( F e r g u s ,  1964).  O f t e n t h e compost becomes w h i t e n e d b y the d e v e l o p m e n t of t h e r m o philic actinomycetes ( " f i r e - f a n g " )  (Renoux-Blondeau,  1959).  However,  o t h e r w o r k e r s h a v e r e p o r t e d the p r e s e n c e of molds in t h e compost ( B e l s - K o n i n g et a l , 1962; Pope et a l , 1962). f l o r a of mushroom compost d u r i n g p h a s e II previously (Renoux-Blondeau, h o l d e r , 1961).  T h e mold a n d a c t i n o m y c e t e  has b e e n i n v e s t i g a t e d  1959; F e r g u s ,  1964; T e n d l e r & B u r k -  H o w e v e r , much work remains to be done - the i d e n t i t y  of t h e m i c r o o r g a n i s m s still has to be r e s o l v e d . s t r a t e d that the a c t i n o m y c e t e s , p e r se,  H a y e s (1977) d e m o n -  a r e of little i m p o r t a n c e  e x c e p t to i n d i c a t e that o t h e r e s s e n t i a l c h a n g e s h a v e o c c u r r e d . A t t e m p t s to c o n t r o l a n d d e v e l o p a s p e c i f i c m i c r o f l o r a (Pope et a l , 1962; R e n o u x - B l o n d e a u , 1959) .have so f a r o n l y been p r e l i m i n a r y . V a r i o u s w o r k e r s ( H a y e s , 1970, 1977; Hayes & R a n d l e , 1970) f u m i g a t e d composts with methyl bromide d u r i n g p h a s e II.  T h i s caused a stimul-  ation in n u m b e r s of b a c t e r i a a n d an e n h a n c e d e f f e c t on p r o d u c t i v i t y . T h i s s u g g e s t s a s t r o n g association between b a c t e r i a l a c t i v i t y 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. of 45-55°C.  Selective compost were produced over the range  Recent results by Gerrits (1980) also indicated that the temp  for maximum mushroom yield may be below 50°C.  Furthermore, in p r e -  liminary work by Ross & Harris (1982), it was found that ammonia disappeared 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 enviroment, the thermophilic population declines (Fordyce, 1970) and Agaricus, which is known to produce an antibiotic (Eger, 1962, 1972; Sinden, 1971) becomes dominant. stable.  Other mesophilic actinomycete populations are relatively  However, Fergus (1971) reports that not all molds are inhibited  17.  by A g a r i c u s , 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 p r e 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 d u r i n g 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 i n o c u l a t i o n o f s p a w n , a n d c a n raise t h e t e m p e r a t u r e h i g h e n o u g h to i n j u r e o r kill t h e mycelium ( S i n d e n , 1971).  In a later s t u d y b y  F e r g u s (1982), it was d e m o n s t r a t e d that d u e to t h e poor heat r e s i s t a n c e of most f u n g i - i f f u n g i a r e to grow i n t h e cooled compost a f t e r s p a w n i n g , t h e y must be i n t r o d u c e d into t h e compost at t h e time o f s p a w n i n g o r thereafter.  T r i g i a n o & F e r g u s (1979) d e m o n s t r a t e d t h a t most o f t h e  fungi d u r i n g spawn-run have the capability of utilizing cellulose, s t a r c h , and  l i p i d s as food s o u r c e s from t h e compost ( t h r o u g h  cellular enzymes).  T h e r e is e v i d e n c e  capable of d e g r a d i n g  lignin.  production of e x t r a -  that many o f these f u n g i may be  F u r t h e r studies a r e needed on the  e n z y m a t i c abilities o f compost f u n g i so t h e i r role i n mushroom c u l t u r e c o u l d be more c l e a r l y u n d e r s t o o d . E x t r a c e l l u l a r p o l y s a c c h a r i d e s a r e s e c r e t e d i n t h e form o f slime l a y e r s o r c a p s u l e s o n t h e e x t e r i o r o f b a c t e r i a l a n d many f u n g a l cells d u r i n g growth.  E d d y (1976) showed that t h e s e l e c t i v i t y o f t h e mushroom compost  f o r mushroom mycelial g r o w t h was c l o s e l y r e l a t e d to t h e composition slime.  of the  E d d y & J a c o b s (1976) a n d S t a n e k (1972) d e m o n s t r a t e d that  b a c t e r i a l p o l y s a c c h a r i d e s were 2-9 times b e t t e r u t i l i z e d b y t h e mycelium t h a n g l u c o s e alone. C h e m i c a l l y ,  this s u b s t a n c e c o n s i s t e d o f g l u c o s e ,  f r u c t o s e , mannose, u r o n i c a c i d a n d n i t r o g e n (0.26%) ( H a y e s , 1977). It was s u g g e s t e d that o t h e r components o f t h e b a c t e r i a l cell may also h a v e important can  roles  to p l a y a n d it is i n t e r e s t i n g to note that A.  synthesize the necessary  cell walls ( T u r n e r , The  bisporus  enzymes to d e g r a d e f u n g a l a n d b a c t e r i a l  1977).  f e r m e n t e d s u b s t r a t e from a S t r e p t o m y c e s s p . a n d a Pseudomonas s p .  19.  w e r e u s e d b y S t a n e k (1972) as a medium t o c u l t i v a t e A.  bisporus.  T h e a e r a t e d s u b s t r a t e from t h e S t r e p t o m y c e s s p . s u p p o r t e d t h e g r o w t h of t h e A g a r i c u s m y c e l i u m , b u t a l l o w e d c o n t a m i n a t i o n ; o n t h e o t h e r h a n d , t h e s u b s t r a t e from t h e Pseudomonas s u p p o r t e d l i t t l e g r o w t h b u t d i d n o t allow c o n t a m i n a t i o n . When t h e two s u b s t r a t e s w e r e m i x e d , c o n t a m i n a t i o n did  not o c c u r a n d i n c r e a s e d m y c e l i a l g r o w t h was e v i d e n t . S t u d i e s b y S t a n e k & Z a t e c k a (1967) a n d T r e s c h o w ( 1 9 4 2 ) , h a v e s h o w n  t h a t most s t r a i n s o f t h e r m o p h i l i c c e l l u l o s e d e c o m p o s i n g a c t i n o m y c e t e s produced pantothenic a n d nicotinic acids, biotin, thiamin, and vitamin Bg. T h e s e s u b s t a n c e s s t i m u l a t e d t h e g r o w t h o f t h e mushroom m y c e l i a . T h e B v i t a m i n s w e r e also formed b y t h e r m o t o l e r a n t f u n g i o f t h e g e n u s Humicola. G r a b b e (1969) f o u n d t h a t h u m i c a c i d s f o r m e d b y m i c r o o r g a n i s m s i n compost w e r e a p o o r s o u r c e o f n u t r i e n t s f o r mushroom m y c e l i u m .  However,  amino a c i d s p r o d u c e d b y v a r i o u s t h e r m o p h i l i c a c t i n o m y c e t e s ( e g . tomyces  thermovulgaris)  Strep-  c o u l d b e u t i l i z e d b y t h e mushroom as a s o u r c e o f  n i t r o g e n ( B o h u s , 1959; P a r k , 1971).  It i s q u i t e e v i d e n t t h a t t h e e x a c t r o l e  of b a c t e r i a i n t h e n u t r i t i o n o f t h e c r o p i s not c o m p l e t e l y c l e a r ( F l e t c h e r , 1979).  F u r t h e r research is o b v i o u s l y needed to establish the precise  c o n t r i b u t i o n o f t h e m i c r o b e t o t h e l i f e c y c l e o f t h e c u l t i v a t e d mushroom. T e n to f o u r t e e n d a y s a f t e r s p a w n i n g , t h e s p a w n w i l l h a v e g r o w n f u l l y t h r o u g h t h e compost a n d w i l l a p p e a r as a g r e y i s h w h i t e g r o w t h o v e r t h e s u r f a c e o f t h e compost ( H a y e s & W r i g h t , 1979). time to l o w e r t h e t e m p e r a t u r e a n d a p p l y t h e c a s i n g s o i l .  T h i s is t h e  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 mushroom (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.  T h e 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. Pseudomonas  putida,  On the other hand. Wood (1976) found that 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» and volatile organic 2  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, C O , and ethylene (Kurtzman, 1979).  Hayes (1972) found that some hemeproteins and E D T A 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 of microbial activity.  2  gradient, and 5) action  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.  f o r e x a m p l e , a n t i b i o t i c s a n d b r e w i n g ; i n w h i c h p a t h o g e n s may be more r e a d i l y e x c l u d e d ( H a y e s , 1974; H a y e s & N a i r , 1975). S u c c e s s f u l e s t a b l i s h m e n t o f A. bisporus  is governed b y factors  w h i c h c o n t r o l t h e e c o l o g y o f t h e two s u b s t r a t e s , u s e d i n c u l t i v a t i o n . T e m p e r a t u r e a n d a e r a t i o n a r e t h e main p h y s i c a l v a r i a b l e s .  This is  u l t i m a t e l y l i n k e d to t h e a c t i v i t y o f m i c r o o r g a n i s m s w h i c h g e n e r a l l y o p e r a t e b y a f f e c t i n g t h e a v a i l a b i l i t y o f n u t r i e n t s ( H a y e s & N a i r , 1975).  A  d e g r e e o f c h e m i c a l c o n t r o l is a c h i e v e d b y p r o v i d i n g t h e c o r r e c t n u t r i e n t s in t h e r e q u i r e d p r o p o r t i o n s t o o b t a i n t h e n e c e s s a r y s u c c e s s i o n o f microorganisms. T h e c a s i n g l a y e r s h o u l d t h e r e f o r e be c o n s i d e r e d as a s u b s t r a t e w h i c h not o n l y s u p p o r t s t h e mushroom b u t also an a s s o c i a t e d m i c r o f l o r a , w h i c h p o s s i b l y b e n e f i t s t h e mushroom a n d s u p p o r t s i t s g r o w t h t h r o u g h t h e i m p o r t a n t t r a n s i t i o n from m y c e l i a l g r o w t h to a. f r u i t b o d y ( H a y e s & W r i g h t , 1979).  Attempts i n the f u t u r e should take into account  the possibilites of biological selection of microorganisms essential f o r s p o r o p h o r e p r o d u c t i o n ( N a i r et a l , 1974).  24.  5.  Fruiting From t h e d e v e l o p e d m y c e l i a l a g g r e g a t e s , p r i m o r d i a o r  pinheads develop, a p r o p o r t i o n o f 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 ( C h a n g & H a y e s , 1978).  T h e mushrooms a p p e a r  in  " b r e a k s " o r f l u s h e s a n d s h o u l d be r e a d y f o r p i c k i n g a b o u t lOd a f t e r  the  p i n h e a d s f i r s t a p p e a r ( B u r d e n & P e t e r s o n , 1972; T o l e m a n , 1979).  M u s h r o o m s a r e t h e n h a r v e s t e d from t h e b e d s o r t r a y s f o r a p e r i o d w h i c h r a n g e s from 28-60d d e p e n d i n g o n t h e p r a c t i c e s o f t h e i n d i v i d u a l g r o w e r ( I n g r a t t a , 1980).  T h e total h a r v e s t d e c r e a s e s f o l l o w i n g t h e t h i r d  f l u s h ( H a y e s & W r i g h t , 1979; V e d d e r , 1978) - r e l a t i n g to t h e d e p l e t i o n o f n u t r i e n t s a n d d e c l i n e o f p H ( C e r r i t s , 1965).  T h e r e is v i r t u a l l y no  k n o w l e d g e o f t h e mechanisms c o n t r o l l i n g t h e s e q u e n t i a l f l u s h i n g o f m u s h r o o m s ( H a y e s 6 N a i r , 1975). A f t e r approximately the fifth break, mesophilic bacteria and known pathogens of A g a r i c u s a r e able to colonize t h e s u b s t r a t e .  A t this  p o i n t , t h e p r o d u c t i o n o f mushrooms becomes uneconomical a n d t h e s p e n t compost i s d i s c a r d e d .  O n many l a r g e r i n t e n s i v e u n i t s , t h e  d i s p o s a l o f s p e n t o r u s e d compost f r e q u e n t l y poses p r o b l e m s .  Recent  s t u d i e s h a v e i n d i c a t e d t h a t m i c r o o r g a n i s m s i n s p e n t compost h a v e a d i r e c t role i n t h e e t i o l o g y o f mushroom w o r k e r ' s l u n g d i s e a s e ( an e x t r i n s i c allergic alveolitis possibly caused b y inhalation of spores of thermophilic a c t i n o m y c e t e s , mushroom v i r u s e s , o r c h e m i c a l s p r e s e n t i n compost) ( K l e y n S W e t z l e r , 1981; K l e y n et a l , 1981). is b e i n g g i v e n  Therefore increasing attention  to s y s t e m s of r e c y c l i n g / r e p l e n i s h i n g t h e n u t r i e n t s w h i c h  h a v e been u t i l i z e d b y t h e mushroom so t h a t t h e y a r e r e u s e d i n t h e  succeeding cycle (eg. hydroponics). Urayama (1961) demonstrated that by spraying Bacillus psilocybe, onto mushroom beds, mycelial density and total production of the fruitbodies 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. workers (Park S Agnihotri, 1969; Park, 1970; Renoux-Blondeaux,  Other  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 o f t h e main r e a s o n s f o r t h e i n v a s i o n o f Chaetomium i n t o m u s h room b e d s i s t h a t some ammonia is left i n t h e compost a f t e r t h e p h a s e II c o o k o u t ( p e a k - h e a t ) o r i s f o r m e d anew ( B e l s - K o n i n g et a l , H a y e s , 1977; R e t t e w , 1948; V e d d e r , 1978). b e d s may  1962;  E x c e s s i v e ammonia i n t h e  be d u e to low amounts o f g y p s u m i n t h e compost d u r i n g  p h a s e I ( C e r r i t s , 1977); p e a k - h e a t i n g f o r too s h o r t o r too l o n g a time, or at too h i g h t e m p e r a t u r e ( a b o v e 55°C) ( K n e e b o n e S M e r e k , 1959; L a m b e r t S M e r e k , 1959; L a m b e r t , 1953; S i n d e n , 1955) a n d h u m i d i t y ; or b y too m u c h c o m p a c t i o n a n d m o i s t u r e ( i e . l a c k o f f r e s h air) 1972; H a y e s S W r i g h t , 1979; S c h i s l e r , 1980; V e d d e r , 1978). more, h i g h c o n c e n t r a t i o n s o f C 0 the  2  (Atkins, Further-  i n t h e compost seems to promote  g e r m i n a t i o n of s p o r e s o f O l i v e g r e e n mold ( C h a n g & H a y e s ,  1978).  Restriction of the airflow (causing anaerobic decomposition) produces as o f y e t u n k n o w n c o m p o u n d s h a z a r d o u s to t h e mushroom b u t r e a d i l y a c c e s i b l e to C h a e t o m i u m ( S c h i s l e r , 1980; S i n d e n , 1971). Excessive or great variations in temperature (especially d u r i n g p h a s e II) p r o d u c e s u n w a n t e d c o n v e r s i o n s i n t h e compost ( i e . p r o t e i n s are  b r o k e n d o w n w h i c h c a n be a s s i m i l a t e d b y O l i v e g r e e n mold  (Lambert,  1953), a n d i t is q u i t e p l a u s i b l e t h a t a n h y d r o u s ammonia w i l l be p r o d u c e d from t h e h i g h e r n i t r o g e n c o m p o u n d s a l r e a d y f o r m e d 1978).  V a r i a t i o n s i n t e m p e r a t u r e may  (Vedder,  o c c u r w h e n t h e g r o w e r a t t e m p t s to  s u p p l e m e n t t h e r m o g e n e s i s w i t h a r t i f i c i a l heat s u c h as l i v e steam.  Too  moist a mushroom b e d will also c a u s e c o n v e r s i o n s to be p u s h e d i n t h e w r o n g d i r e c t i o n , b e c a u s e s u f f i c i e n t amounts o f o x y g e n a r e u n a b l e to p e n e t r a t e t h e compost.  In o t h e r w o r d s , w h e n a n y o f t h e s e c o n d i t i o n s  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 spawnrun (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 t u r n i n g .  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).  musty odor (Vedder,  The mold gives the manure a typical dank or 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 mushrooms will appear at all. Furthermore, spawn growth is retarded or fails altogether (Stanek, (1979)  1967).  As indicated by Trigiano & Fergus  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. O l i v e g r e e n mold i s also f o u n d o n b e d s w h i c h h a v e b e e n s u p p l e m e n t e d w i t h n u t r i e n t s a f t e r p h a s e II ( S i n d e n & S c h i s l e r , 1962) o r d u r i n g c o m p o s t i n g ( H a y e s , 1977; V e d d e r , 1978).  Chaetomium seems to be  the l i m i t i n g f a c t o r ( i n t h e amount o f s u p p l e m e n t a d d e d ) b e c a u s e i t competes f o r t h e s u p p l e m e n t w i t h t h e mushroom mycelium - w i t h t h e slow g r o w i n g mycelium l o s i n g o u t . C h a e t o m i u m is also a s e r i o u s p a t h o g e n o f c o t t o n , s o y b e a n and f l o w e r c r o p s ( K a n w a r et a l , 1979; N i k , 1980).  sun-  It c a u s e s b i o l o g i c a l  damage to wool f i b e r s ( S a n k o v et a l , 1972), a n d d e t e r i o r a t e s s y n t h e t i c r e s i n s s u c h as u r e t h a n e r u b b e r ( T a k e y o s h i et a l , 1971).  Chaetomium  is c u r r e n t l y k n o w n to p r o d u c e a w i d e v a r i e t y o f d i f f e r e n t t o x i c metabolites  ( i e . m y c o t o x i n s ) ( B r e w e r et a l , 1970; B r e w e r & T a y l o r ,  1978; S e k i t a et a l , 1981) s u c h as s t e r i g m a t o c y s t i n to a f l a t o x i n s ) , o o s p o r e i n , globosins.  (possible  precursor  cochliodinol, chaetomin, and the chaeto-  T h e l a t t e r two p r o d u c t s h a v e b e e n t h e r e s p o n s i b l e a g e n t s  i n s e v e r a l m y c o t o x i c o s e s . T h i s o r g a n i s m (Chaetomium) has also been i m p l i c a t e d i n at least one p a t i e n t d e a t h i n t h e U n i t e d S t a t e s c o m m u n i c a t i o n ; D r . M.G. S t a t e U n i v e r s i t y ) .*  R i n a l d i , Dept. of Microbiology,  (personal  Montana  C.  BIOLOGICAL C O N T R O L  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; B a k e r £ C o o k , 1974; B a k e r £ S n y d e r , 1965; G a r r e t t , 1955, 1965; H u s s e y , 1969; H e n i s £ C h e t , 1975).  T h e extreme difficulty of  i s o l a t i n g , c o r r e l a t i n g , a n d u n d e r s t a n d i n g t h e many f a c t o r s t h a t i n f l u e n c e m i c r o b i a l a c t i v i t e s a r o u n d root s y s t e m s a n d o n p l a n t s u r f a c e s has impeded t h e d e v e l o p m e n t o f b i o l o g i c a l c o n t r o l p r a c t i c e s o f commercial b e n e f i t ( G a r r e t t , 1955; S c r o t h £ H a n c o c k ,  1981).  Thus, despite the  many d e c a d e s o f r e s e a r c h , t h e r e a r e o n l y two c a s e s w h e r e a b i o l o g i c a l c o n t r o l a g e n t h a s been r e g i s t e r e d f o r u s e b y a g o v e r n m e n t  agency and  is c o m m e r c i a l l y u s e d i n N o r t h A m e r i c a ( b i o l o g i c a l c o n t r o l o f i n s e c t s i s excepted). T h e p r o b a b l e mechanisms o f b i o l o g i c a l c o n t r o l a r e t h a t o f a l i v i n g organism acting d i r e c t l y on the pathogen (antagonism) o r t h r o u g h t h e i n t e r m e d i a t e a g e n c y o f t h e h o s t ( B a k e r , 1968).  T h e two main c a t e g o r i e s  of a n t a g o n i s m a r e a n t i b i o s i s a n d c o m p e t i t i o n ( P a r k , 1960). (Scroth £ Hancock,  1981) i s d e f i n e d as a n i n t e r a c t i o n  Antibiosis  between  o r g a n i s m s w h e r e b y a metabolic a g e n t p r o d u c e d b y o n e o r g a n i s m h a s a harmful effect on the other.  U s u a l l y t h i s d e f i n i t i o n e x c l u d e s common  metabolic p r o d u c t s , s u c h as c a r b o x y l i c a c i d s , e t h a n o l , a n d C 0 . 2  This  t o p i c h a s also been r e v i e w e d r a t h e r e x t e n s i v e l y i n t h e l i t e r a t u r e ( B r i a n , 1957; G a r r e t t , 1956; J a c k s o n , 1965).  Production of antibiotics on  g r o w t h media i s common among soil f u n g i , a c t i n o m y c e t e s a n d b a c i l l i .  How-  e v e r , the function of antibiosis in biological control, their formation and e c o l o g i c a l s i g n i f i c a n c e i n common soil h a v e i n d u c e d a c o n s i d e r a b l e d e b a t e ( B a k e r £ C o o k , 1974; J a c k s o n , 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). T h e 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 mushroom 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 (mechanism 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 d r y 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 IO / I was an effective control of Verticillium. 6  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 b y 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 MATERIALS  C u l t u r e s o f Agaricus bisporus  w e r e r e c e i v e d from t h e F r a s e r V a l l e y  M u s h r o o m G r o w e r s Co-op ( L a n g l e y , B.C.)  a n d m a i n t a i n e d o n Potato  d e x t r o s e a g a r ( D i f c o ) at 25°C and 3°C.  C u l t u r e s o f Chaetomium olivaceum w e r e r e c e i v e d from D r . L . C . S c h i s l e r , Dept. of Plant Pathology, P e n n s y l v a n i a State U n i v e r s i t y ; C. olivaceum  was also m a i n t a i n e d o n Potato d e x t r o s e a g a r at 25°C a n d  3°C.  I_.  SELECTION OF THERMOPHILES R e p r e s e n t a t i v e (40g) samples o f commercial compost w h i c h  had  been p a s s e d t h r o u g h t h e i n i t i a l s t a n d a r d mushroom c o m p o s t i n g s t a g e ( p h a s e I) p r i o r to d e l i v e r y to t h e g r o w e r f o r p a s t e u r i z a t i o n ( p h a s e II) w e r e r e c e i v e d o n a w e e k l y b a s i s from t h e F r a s e r V a l l e y M u s h r o o m Growers Compost D i v i s i o n .  T h e s e samples w e r e immediately i n c u b a t e d u p o n  r e c e i p t f o r two d a y s at 55°C u n d e r a e r o b i c c o n d i t i o n s a n d h i g h h u m i d i t y ( F i s h e r Isotemp i n c u b a t o r ) . T h i s i n c u b a t i o n p r o v i d e d a c o n t r o l l e d d u p l i c a t i o n o f t h e f i n a l t h e r m o p h i l i c t r e a t m e n t g i v e n b y t h e mushroom g r o w e r b e f o r e i n o c u l a t i o n w i t h mushroom s p a w n ( R o s s & H a r r i s , 1982). F o r b a c t e r i o l o g i c a l a n a l y s i s , lOg o f t h e t h e r m o p h i l i c compost was w i t h 90ml o f s t e r i l e d i s t i l l e d w a t e r a n d t h e sample  mixed  kneaded thoroughly in  o r d e r to p l a c e s u f f i c i e n t m i c r o f l o r a i n t o s u s p e n s i o n .  Thereafter, tenfold  38.  dilutions using sterile distilled water were prepared to a 10 -2 Aliquots of 0.1ml from the 10  dilution.  -7 to 10  dilutions were spread plated in  duplicate on T S Y 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 T S Y 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 T S Y 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. B e c a u s e o f the p o o r r e s u l t s a s s o c i a t e d w i t h t h i s method (see S D i s c u s s i o n s e c t i o n ) a r e v i s e d p r o c e d u r e was  Results  d e s i g n e d (see (b) ).  S y n t h e t i c compost medium The of:  CaC0  MgS0  2  0.7g  4  0.2g  4  KCI  O.lg  FeS0  4  • 7H 0  lOmg  2  distilled water  1000ml ( f i n a l pH  T h i s n u t r i e n t s s o l u t i o n was 15psi.  composed  1. Og  3  (NH ) HP0 4  s y n t h e t i c compost medium was  7.0)  t h e n s t e r i l i z e d u s i n g steam f o r I5min at  F i f t e e n ml of the s t e r i l e n u t r i e n t s o l u t i o n was  a d d e d to 5.5g  s t e r i l e b a l l - m i l l e d wheat s t r a w ( i n a g l a s s p e t r i d i s h ) a n d compost medium.  The  n u t r i e n t was  called synthetic  c a r e f u l l y a d d e d at a l e v e l j u s t  s u f f i c i e n t to e v e n l y moisten t h e v e r y d r y  straw.  of  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 T S Y 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 T S Y agar and later stored on T S Y agar slants at 3°C.  The 45 previously isolated colonies (from Section a)  were also tested in this way.  4 0 g  phase  1  compost 4 B h ;  IO g  +  S Om l  s .d . w .  *  serial  dilution  55°C  -7  - a  I O  t o  O.I m l spread  plate  o n T S Y agar 16-SO  spray  with C  of  5 5 '  suspension of  olivaceum 25°  examine  h;  C  daily f o r z o n e s inhibition  Figure 2. Selection of thermophiles for activity against Chaetomium olivaceum.  II.  I D E N T I F I C A T I O N OF T H E R M O P H I L E S  1_. M i c r o s c o p i c a p p e a r a n c e a)  Gram stain: Young cultures of the thermophiles previously grown  on T S Y a g a r (12-20h) w e r e G r a m s t a i n e d a n d o b s e r v e d f o r s i z e (micrometer) a n d s h a p e o f c e l l s , m o r p h o l o g y a n d p a r t i c u l a r g r o u p i n g s , a n d p r e s e n c e / l o c a t i o n o f s p o r e s ( u s i n g oil immersion b)  microscopy).  Spore stain: C u l t u r e s w e r e s t a i n e d a c c o r d i n g to t h e method o f  Dorner  ( D o e t s c h , 1981) a n d o b s e r v e d f o r t h e l o c a t i o n a n d n a t u r e o f  s p o r e b o d i e s ( a n y s w e l l i n g o f t h e s p o r a n g i a was also n o t e d ) .  Spores  w e r e o b s e r v e d to s t a i n - r e d a g a i n s t a c o l o r l e s s b a c t e r i a l c e l l .  2. M a c r o s c o p i c a p p e a r a n c e T h e r m o p h i l e s w e r e o b s e r v e d f o r c o l o n y morph o l o g y s u c h as s i z e , s h a p e , m a r g i n , e l e v a t i o n , p i g m e n t a t i o n , e t c . , on T S Y and P D A  agars.  43.  3.  Motility The motility of young cultures of each thermophile was  determined b y : a)  direct microscopic observation from wet mounts of the organisms (Smibert & Krieg, 1981).  b)  use of semisolid media (0.7% agar + T S Y 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 & G e r 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 observation. 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  Systems) and catalyst.  Microbiology  44.  5.  Maximum a n d minimum t e m p e r a t u r e s o f g r o w t h C u l t u r e s were inoculated  into T S Y b r o t h a n d i n c u b a t e d i n d u p l i c a t e a t : a)  f o r maximum t e m p e r a t u r e  d e t e r m i n a t i o n - 55, 60, 65, 70, a n d 75°C i n a w a t e r b a t h ( B l u e M, I l l i n o i s ) . G r o w t h o f t h e c u l t u r e s w e r e d e t e r m i n e d a f t e r 3d ( G o r d o n et a l , 1973) b y the presence o r absence o f t u r b i d i t y .  D u r i n g incubation, the water  level o f t h e b a t h was c a r e f u l l y m a i n t a i n e d . b) d e t e r m i n a t i o n - 15, 25, 35, 45, a n d 55°C.  f o r minimum t e m p e r a t u r e  F i f t e e n c e n t i g r a d e was t h e  lowest t e m p e r a t u r e t h a t c o u l d b e m a i n t a i n e d a c c u r a t e l y .  G r o w t h was  r e c o r d e d a f t e r 5d at t e m p e r a t u r e s b e t w e e n 35-55°C a n d a f t e r 21d at 15°C ( G o r d o n et a l , 1973) .  6.  Biochemical reactions The following biochemical tests were performed  on t h e t h e r m o p h i l e s : a)  Catalase test:  ( S m i b e r t & K r i e g , 1981).  b)  Methyl r e d test:  c)  Hydrolysis of urea:  d)  Hydrolysis of starch:  e)  Production of indole:  ( S m i b e r t & K r i e g , 1981). ( S m i b e r t & K r i e g , 1981). ( G o r d o n et a l , 1973). ( S m i b e r t & 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 ( B B L ) . ii)  method of Gordon et al (1973),  utilizing a revised VP medium. g)  Growth in NaCl:  containing 0, 5, 7, and 10% (  Tubes of nutrient broth  /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). h)  Growth on Mannitol salt agar:  i)  Resistance to lysozyme:  J)  Utilization of sodium citrate:  k)  Utilization of sodium propionate:  I)  Reduction of nitrate to nitrite:  m)  Deamination of phenylalanine:  (Smibert &  Krieg, 1981). (Gordon et al, 1973). (Gordon et al, 1973). (Gordon et  al, 1973). (Gordon et al, 1973). (Gordon et al, 1973).  46.  n)  Decomposition o f t y r o s i n e :  o)  G r o w t h at p H 5.7:  ( C o r d o n et a l , 1973).  T S Y b r o t h was p r e p a r e d  a c c o r d i n g to t h e m a n u f a c t u r e r a n d t h e p H a d j u s t e d to 5.7 w i t h IN HC1. Media was f i l t e r s t e r i l i z e d (0.45 um m i l l i p o r e f i l t e r ) a n d a s e p t i c a l l y d i s t r i b u t e d into sterile test tubes. d u p l i c a t e a n d i n c u b a t e d at 55°C.  C u l t u r e s were inoculated i n G r o w t h was r e c o r d e d at 7 a n d I4d o f  incubation.  p)  G r o w t h i n sodium azide: T u b e s of azide d e x t r o s e  b r o t h w e r e p r e p a r e d b y two m e t h o d s ; i)  a c c o r d i n g to t h e d i r e c t i o n s o f  G o r d o n et a l (1973). ii)  azide d e x t r o s e b r o t h ( F i s h e r Gram-  P a c , P i t t s b u r g h ) , p r e p a r e d a c c o r d i n g to m a n u f a c t u r e r ' s  directions.  B o t h methods r e s u l t i n a f i n a l c o n c e n t r a t i o n o f 0.02% a z i d e i n e a c h t u b e . C u l t u r e s w e r e i n o c u l a t e d i n d u p l i c a t e i n t o b o t h t y p e s o f medium a n d i n c u b a t e d at 55°C.  G r o w t h was o b s e r v e d a f t e r 7 a n d I4d o f i n c u b a t i o n .  q) of D  A c i d from c a r b o h y d r a t e s :  10% a q u e o u s s o l u t i o n s  (-K-)- g l u c o s e ( A m a c h e m ) , L (+)- a r a b i n o s e , D (+)- x y l o s e , a n d D ( - ) -  mannitol w e r e f i l t e r s t e r i l i z e d (0.45um m i l l i p o r e ) a n d a s e p t i c a l l y a d d e d to p h e n o l r e d b r o t h b a s e ( D i f c o ) to y i e l d a f i n a l c o n c e n t r a t i o n o f 1% s u g a r i n each tube. C u l t u r e s w e r e i n o c u l a t e d i n t o s u g a r s i n d u p l i c a t e a n d i n c u b a t e d at 55°C. A c i d p r o d u c t i o n was r e c o r d e d at 2, 7, a n d I4d.  47.  r)  Production of dihydroxyacetone:  s)  Milk reactions:  (Cordon et al,  1973). 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 d r y 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.  ML  U S E O F B A C I L L U S A O G IN M U S H R O O M  A.  C o n v e n t i o n a l P r o d u c t i o n o f Agaricus  CULTIVATION  bisporus  500g o f s t a n d a r d p h a s e II mushroom compost was s p a w n e d w i t h 9.0g o f Agaricus  bisporus  s p a w n g r a i n s ( o b t a i n e d from F r a s e r V a l l e y  Mushroom G r o w e r s Association) a n d placed in plastic g r o w i n g t r a y s (8 x 15 x 23cm).  Elemental a n a l y s i s o f t h e compost u s e d  (Canadian  M i c r o a n a l y t i c a l L a b o r a t o r i e s , V a n c o u v e r ) d e m o n s t r a t e d i t to c o n t a i n 46.7% C a r b o n a n d 2.14% N i t r o g e n ( a v g . o f 3-50g s a m p l e s ) .  T h i s s p a w n e d compost  was t h e n s u b j e c t e d to v a r i o u s t r e a t m e n t s :  a) c o n t r o l - no f u r t h e r a d d i t i o n to t h e compost b e d ; b) 1.00ml o f a 10^/ml t h e r m o p h i l i c B a c i l l u s A O G c u l t u r e g r o w n i n T S Y b r o t h f o r 96h at 55°C (New B r u n s w i c k P s y c h r o t h e r m s h a k e r ) was a d d e d ; c) 100ml o f a 10^/ml t h e r m o p h i l i c B a c i l l u s A O G c u l t u r e g r o w n i n T S Y b r o t h f o r 96h at 55°C was a d d e d ; f o l l o w e d b y s p r a y i n g 2.0ml o f a Chaetomium  olivaceum  spore suspension on top o f  the bed ; o r , d ) 2.0ml o f a C. olivaceum the b e d only.  spore suspension sprayed on top of  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 limestone (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» levels at a minimum (ie. C 0 2  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_. H y d r o p o n i c P r o d u c t i o n o f Agaricus  bisporus  w 2% ( /v) Malt e x t r a c t ( D i f c o ) , c o n t a i n i n g 2% CaCC» a n d 0.4% 3  Y e a s t e x t r a c t ( p H 7.0) o r a l i q u i d compost s o l u t i o n (see below) w e r e c h o s e n as t h e l i q u i d n u t r i e n t s f o r mushroom h y d r o p o n i c c u l t u r e . L i q u i d compost: L i q u i d compost was p r e p a r e d b y a d d i n g 5L o f d i s t i l l e d w a t e r to I k g wet w e i g h t o f mushroom compost.  T h r e e lOOg  samples o f t h e compost w e r e o v e n d r i e d at 80°C f o r 48h to d e t e r m i n e total s o l i d s p r e s e n t .  T h e m i x t u r e was a g i t a t e d v i g o r o u s l y (584rpm) i n  a t h e r m o p h i l i c w a s t e f e r m e n t e r ' ( F i g u r e 3) w i t h a i r b e i n g p u m p e d i n at 4800 ml/min to m a i n t a i n a e r o b i c c o n d i t i o n s .  T h e compost e x t r a c t was t h e n  c o l l e c t e d b y f i l t r a t i o n t h r o u g h c h e e s e c l o t h a n d s t e r i l i z e d u s i n g steam at 15psi f o r I5min.  T h e r e s u l t i n g s u b s t r a t e was c a l l e d l i q u i d compost.  Final  p H o f t h i s medium was 7.2.  T o t h i s l i q u i d compost a n d to t h e 2% Malt e x t r a c t was i n o c u l a t e d t h e Bacillus AOG culture.  T h e s e l i q u i d s u b s t r a t e s w e r e t h e n i n c u b a t e d at 55°C 4  f o r 96h to y i e l d a f i n a l c o n c e n t r a t i o n o f 10 count on T S Y agar) .  cells/ml (determined b y plate  D u r i n g t h e a b o v e 96h i n c u b a t i o n , 2ml samples w e r e  a s e p t i c a l l y r e m o v e d on a d a i l y b a s i s from t h e 2% Malt e x t r a c t medium f o r total c a r b o h y d r a t e a n a l y s i s ( s e e b e l o w ) .  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/  t r a y ) , representative trays with or without the Bacillus were sprayed with 2.0ml of a C. olivaceum 12d.  spore suspension and incubated at 25°C for  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 respectively) 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 B A C I L L U S AOG  A.  Determination of a pH Change a)  Bacillus AOG was streaked once across 6 T S Y 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.  T h e pH of 4 un-  inoculated (control) T S Y plates was also measured.  b)  10ml of T S Y broth were placed in 18 x 150mm test tubes  and sterilzed for 15min at I5psi.  T h e 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)  T S Y broth:  Bacillus AOG was inoculated  into 100ml of T S Y 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.  T h e 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 T S Y 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; s t a r c h , 0.5g;  C a C 0 , 0.5g; 3  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 T S Y agar plates.  Plates  were sprayed with a spore suspension of C. olivaceum, incubated for 5d at 25°C and examined daily for zones of inhibition. 0  56.  c)  T S Y 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. with C. olivaceum  Following incubation, plates were sprayed  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 uninoculated T S Y 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 i s c s w e r e t h e n p l a c e d onto T S Y a g a r p l a t e s , s p r a y e d w i t h a s p o r e s u s p e n s i o n o f O l i v e g r e e n mold, a n d i n c u b a t e d f o r 4-5d at 25°C ( e x a m i n e d d a i l y f o r zones o f i n h i b i t i o n ) .  ii)  Mesophilic conditions: Since t h e T S Y agar plates showing  i n h i b i t i o n a g a i n s t O l i v e g r e e n mold ( b y B a c i l l u s A O G ) h a d b e e n i n c u b a t e d at m e s o p h i l i c t e m p e r a t u r e s (ie. 25°C), t h e i n h i b i t o r may o n l y b e p r o d u c e d b y B a c i l l u s A O G at t h i s l o w e r t e m p e r a t u r e r a n g e . H e n c e , B a c i l l u s A O G was i n o c u l a t e d i n t o 100ml o f T S Y b r o t h a n d i n c u b a t e d f o r 2, 4, 6, a n d 8d at 25, 37, a n d 55°C i n a s h a k e r w a t e r b a t h . C u l t u r e s w e r e t h e n c e n t r i f u g e d at 8000rpm (10,400 x G; G S A r o t o r ) i n a S o r v a l l R C 2 - B (0°C) f o r 35min a n d f i l t e r e d t h r o u g h a 0.22um m i l l i p o r e . T h e f i l t r a t e was t h e n f r e e z e - d r i e d a n d r e c o n s t i t u t e d i n e i t h e r 1ml o f c o l d s t e r i l e d i s t i l l e d w a t e r o r 1ml o f n - b u t a n o l . Samples (0.1ml) w e r e s o a k e d onto f i l t e r p a p e r d i s c s , p l a c e d onto T S Y a g a r p l a t e s a n d s p r a y e d with  C.  olivaceum.  P l a t e s w e r e e x a m i n e d d a i l y f o r zones o f i n h i b i t i o n a f t e r  4d i n c u b a t i o n at 25°C.  58.  iii) Evaporation method: Cultures of Bacillus AOG were incubated in T S Y broth for 7d at 2 5 ° C , 150rpm (New Brunswick shaker).  Psychrotherm  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).  o r , 2)  frozen at -15°C for 48h, and 3 months (Viking  freezer).  Following treatments, the pipette contents were placed onto filter paper discs on T S Y 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 T S Y agar ( 2 5 ° C ) .  60.  E_.  Solvent Solubility F r e e z e - d r i e d c u l t u r e s o f 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 w a t e r as o u t l i n e d p r e v i o u s l y . solvent (water, n-butanol, n-propanol,  One  ml of a p a r t i c u l a r  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 tetrachloride, or cyclohexanol) t h e n a d d e d to t h e 1ml o f B a c i l l u s AOG  e x t r a c t . The  was  solvents were all of  r e a g e n t , A . C . S . o r U.S.P. g r a d e . Samples w e r e t h e n w o r t e x e d v i g o r o u s l y f o r 1min a n d then, c e n t r i f u g e d at 3000rpm i n a SS-34 r o t o r (1085 x G; c e n t r i f u g a t i o n , the pH e x t r a c t e d l a y e r and  was  0.1ml  a sterile filter paper disc.  0°C)  f o r 15min.  d e t e r m i n e d ( F i s h e r pH  Following  paper) of the solvent  (of s o l v e n t e x t r a c t e d l a y e r ) was  placed onto  T h e s e d i s c s w e r e let s t a n d i n a s t e r i l e p e t r i  d i s h f o r 12h at room t e m p e r a t u r e i n o r d e r to e v a p o r a t e e x c e s s s o l v e n t . 0.1ml  o f t h e s o l v e n t o n l y was  a l s o a d d e d to a c o n t r o l d i s c .  A l l experiments  were conducted in duplicate. D i s c s w e r e t h e n p l a c e d on a T S Y  agar plate, challenged with Olive  g r e e n mold a n d i n c u b a t e d at 25°C f o r 4d. of inhibition were measured and  recorded.  F o l l o w i n g i n c u b a t i o n , zones  61.  F.  Spectrum of Activity of Antibiotic Bacillus AOG was streaked once across a T S Y agar plate and  incubated for 20h at 5 5 ° C .  Following incubation, various test organisms  (Table 1) were streaked on the same T S Y plate at right angles to the streak of Bacillus A O G .  All test organisms had previously been  grown on T S Y 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. or U . S . P . grade.  The solvents employed were all of reagent, A . C . S ,  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  T a b l e 1. M i c r o o r g a n i s m s t e s t e d f o r s u s c e p t i b i l i t y to a n t i b i o t i c .  Organism  ATCC  Gram negative Serratia marcescens Proteus vulgaris Escherischia coli Klebsiella pneumoniae Enterobacter cloacae  8100 13315 25922 13883 23355  Gram positive Staphylococcus aureus Streptococcus pyogenesStreptococcus lactis Streptococcus cremoris Bacillus subtilis Bacillus megaterium  25923 19615 19435 19257 28281 25848  Yeasts Candida lipolytica Candida utilus Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces pastorianus  8862 9256 7753 7754 2 339  63.  t a n k s w e r e allowed to e q u i l i b r a t e w i t h t h e d e v e l o p i n g s o l v e n t b e f o r e e a c h run.  D e v e l o p m e n t took p l a c e a t room t e m p e r a t u r e .  chromatograms were developed with n-butanol  The preparative  - acetic acid (glacial) -  w a t e r (60:20:20 V / V / V ) f o r a d i s t a n c e o f I5cm ( a p p r o x .  12h). T h e  plates w e r e r e m o v e d from t h e t a n k s , a i r d r i e d , a n d b i o l o g i c a l l y a c t i v e b a n d s (see below) e x t r a c t e d t h r e e times w i t h  n-butanol.  0.5ml o f t h e b i o l o g i c a l l y a c t i v e b a n d s w e r e s t r e a k e d a c r o s s t h e o r i g i n o f s e p a r a t e 20 x 20cm p l a s t i c S i l i c a g e l a n a l y t i c a l p l a t e s (0.2mm t h i c k n e s s ) w i t h no i n d i c a t o r ( M e r c k K i e s e l g e l 60). T h e c h r o m a t o g r a m s w e r e t h e n r u n i n a methanol - c h l o r o f o r m - 17% ammonium  hydroxide  s o l v e n t s y s t e m (40:10:20 V / V / V ) f o r a d i s t a n c e o f 15cm ( a p p r o x .  90  min).at-room t e m p e r a t u r e . R, v a l u e s o f a p p r o p r i a t e b a n d s w e r e c a l c u l a t e d a n d r e c o r d e d .  Location a n d c h 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 c h r o m a t o g r a m s w e r e e x a m i n e d u n d e r b o t h short a n d long wave ultraviolet light (ChromatoVue; Ultraviolet  Products  Inc., San G a b r i e l , C a l i f . ) . ii)  b i o l o g i c a l l y a c t i v e b a n d s - t o locate t h e b i o l o g i c a l l y  a c t i v e b a n d s , 2.5cm o f a T L C p l a t e was l a y e r e d w i t h a t h i n s e c t i o n 0.3cm) o f T S Y a g a r , s p r a y e d 4d.  w i t h C. olivaceum  (approx.  a n d i n c u b a t e d at 25°C f o r  C h r o m a t o g r a m s e c t i o n s w e r e o b s e r v e d d a i l y f o r t h e p r e s e n c e o f zones  of inhibition.  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  T S Y agar, sprayed with Olive green mold, and incubated for 4d at 2 5 ° C . Plates were examined daily for the presence of zones of inhibition. iv)  spray reagents - a)  indantrione monohydrate (Ninhydrin)  Ninhydrin:  0.15g of 1, 2, 3,  (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 T L C 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:  T L C 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 ^ S O ^ in 95ml ethanol.  Chromatograms were then  s p r a y e d , and heated for I5min at 100°C (Stahl, 1969).  65.  d)  Alpha-cy  a t o g r a m s w e r e s p r a y e d w i t h aZpha-cycIodextrin  clodextr'm:  Chrom-  (30% ethanolic solution  o f aZpha-cyclodextrin), a i r - d r i e d , a n d p l a c e d i n a c l o s e d t a n k c o n t a i n i n g iodine vapor.  T h i s spray reagent indicates the presence of straight  c h a i n 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 S p e c t r u m P u r i f i e d samples o f B a n d s I (3.1mg) a n d II (5.0mg) ( T L C ) i n n b u t a n o l w e r e s c a n n e d u n d e r 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 S p e c t r o p h o t o m e t e r ) a n d d a t a was r e c o r d e d . T h e a b s o r p t i v i t y o f t h e samples w e r e also c a l c u l a t e d , u s i n g t h e e q u a t i o n :  A = a  w h e r e "A  b •c  absorbance  a  absorptivity  b  l i g h t p a t h (cm)  c  concentration  (mg/ml)  66. ]_.  Column Chromatography  i)  lon-exchange chromatography: a)  column p r e p a r a t i o n :  A cation  e x c h a n g e r e s i n - A G 50W X-8 w i t h 100-200 mesh s i z e ( B i o r a d ) was hydrated  w i t h d i s t i l l e d d e i o n i z e d w a t e r a n d p o u r e d i n t o a 2.0 x 25cm  glass column. T h i s r e s i n was c o n v e r t e d  to t h e H  f o r m b y w a s h i n g w i t h 150ml o f  +  IN HCI a n d t e s t i n g f o r low p H ( 0 . 5 ) .  T h e c o l u m n was t h e n r i n s e d w i t h  200ml o f d e i o n i z e d w a t e r f o r a f i n a l p H o f 6.5.  b) sample ( B a c i l l u s a n t i b i o t i c ) i n n - b u t a n o l column.  sample e l u t i o n :  10ml o f t h e  was c a r e f u l l y l a y e r e d o n t o t h e  E l u t i o n was p e r f o r m e d w i t h a d i s c o n t i n o u s g r a d i e n t o f 100ml o f  0.01N, 0.02N, 0.05N, 0.08N, a n d 0.1N NH^OH. 1ml/min was m a i n t a i n e d  at room t e m p e r a t u r e .  A c o l u m n flow r a t e o f  Samples w e r e c o l l e c t e d  on a n Isco F r a c t i o n C o l l e c t o r . c)  d e t e c t i o n o f sample:  T h e O.D. o f  all t u b e s w e r e r e a d at U.V. 275um a n d 270um a n d d a t a r e c o r d e d C a r y 210  Spectrophotometer).  (Varian  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) U.V.  sample detection:  Each tube was scanned under  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  B i o l o g i c a l l y a c t i v e b a n d s (from T L C a n a l y s i s ) w e r e s c a n n e d o n a n Aminco-Bowman Spectrophotofluorometer  and spectra  recorded.  K. A m i n o A c i d A n a l y s i s  A m i n o a c i d a n a l y s i s was p e r f o r m e d o n p u r i f i e d samples o f t h e a n t i b i o t i c . F o l l o w i n g e x t r a c t i o n from T L C p l a t e s as p r e v i o u s l y d e s c r i b e d , a p p r o p r i a t e bands (ie. 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 ( l o w e r T L C b a n d , 2.1mg; u p p e r T L C b a n d , 1. 3mg) w e r e t h e n h y d r o l y z e d w i t h p - t o l u e n e s u l f o n i c a c i d i n t h e p r e s e n c e o f 3-(-2  amino-  e t h y l i n d o l e ) f o r 24h at 110°C a c c o r d i n g to t h e method o f L i u & C h a n g (1971). was  B e c a u s e o f v e r y low sample amounts no S H b l o c k i n g o f c y s t e i n e  c o n d u c t e d , a n d o n l y 1ml o f I N N a O H was a d d e d t o samples a f t e r  hydrolysis. The  d i g e s t e d samples w e r e t h e n f i l t e r e d t h r o u g h a n u l t r a f i n e s i n t e r e d  glass filter.  T h e filter had p r e v i o u s l y been r e v e r s e d f l u s h e d with IN  N a O H , d i s t i l l e d w a t e r , a n d n e u t r a l i z e d w i t h I N HC1; a f i n a l r i n s e was c o n d u c t e d with d i s t i l l e d water a n d then a i r d r i e d u s i n g acetone.  0.5ml  o f t h e f i l t e r e d samples w e r e a n a l y z e d o n a P h o e n i x Model 6880 ( P h o e n i x Instruments,  P h i l . ) amino a c i d a n a l y z e r u t i l i z i n g a s i n g l e c o l u m n e l u t i o n  s y s t e m ( D u r r a m C h e m i c a l C o r p . , Palo A l t o , C a l i f . ) . '  69.  R E S U L T S 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  a medium which resembles actual compost substrates (ie. compost medium").  using  "synthetic  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 d i s 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 i g u r e t.  Ultraviolet s p e c t r a of l i g n i n e x t r a c t e d from wheat straw a n d 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 T S Y 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 T S Y plates. Agaricus  bisporus  The organisms capability to support  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 T S Y 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 Figure 5.  d.  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. 1x4) chains.  (Figure 6), occurring generally in singles, pairs, and short 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 aerotolerant Clostridia". The maximum growth temperature of these organisms was observed to  T a b l e 2.  M i c r o s c o p i c o b s e r v a t i o n s a n d colony morphology o f isolated  T h e r m o p h i l e s #1-9  Gram  T h e r m o p h i l e #10  stain  Size - length width spore  thermophiles.  (um) (um)  arrangement  rods ( s i n g l e s , p a i r s short chains)  rods ( s i n g l e s , p a i r s short chains)  2.0 - 4.8 .78 - 1.0  1.7 - 5.31 .78 - .94  s u b t e r m i n a l to terminal  s u b t e r m i n a l to terminal  large  large  A p p e a r a n c e o f colonies TSY  agar  size shape  (3-10mm)  generally  margin  pigmentation  colony u n d e r r e f l e c t e d light t r a n s m i t t e d light elevation  round  r o u n d to i r r e g u l a r  irregular  smooth to i r r e g u l a r (undulate)  green/brown  tan to cream (slight green)  dull translucent  shiny opaque  relatively  flat  other  PDA  (2-12mm)  umbonate/hilly extremely mucoid; some volcano-1 ike structures  agar  pigmentation colony u n d e r r e f l e c t e d  light  transmitted  light  elevation other A g a r slant g r o w t h thermophiles  #7, 9 were o b s e r v e d  dull translucent  relatively dull opaque  flat  umbonate  no mycelium  no mycelium  effuse  to b e Gram  negative.  effuse  F i g u r e 6.  G r a m r e a c t i o n of T h e r m o p h i l e # 10.  Table  3.  P r o p e r t i e s o f isolated  Property  thermophiles.  Thermophiles  #1-9  Thermophile  #10  Motility Anaerobic  growth  T e m p e r a t u r e Maximum Minimum  60 °C  65°C  15°C  15°C  Catalase Voges-Proskauer Methyl-red R e s i s t a n c e to lysozyme G r o w t h i n 10% N a C l : G r o w t h at p H  5.7  G r o w t h i n 0.02% a z i d e A c i d from  glucose arabinose xylose mannose  Hydrolysis of starch Utilization o f u r e a citrate propionate N0 " 3  to N 0 " 2  Production of dihydroxyacetone indole Deamination o f p h e n y l a l a n i n e Decomposition  Litmus  milk  Mannitol salt  of casein tyrosine a l k a l i n e clot reduction  alkaline clot reduction  77.  be 60 to 6 5 ° 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 - 7 5 ° C ; B. coagulans,  55 - 60 (65) ° C ;  B. brevis,  40 - 6 0 ° C ; B. licheniformis,  50 - 5 5 ° 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. grow in acid media.  Ability of isolated thermophiles to  T a b l e 4.  Summary o f p r o p e r t i e s o f known thermophilic Bacillus s p e c i e s .  Property  Thermophile  size - width (u) - l e n g t h (u) Gram stain spore  formation  stearothermophilus  B. coaqulans  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  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 + central/ paracentral; s l i g h t swelling of s p o r a n g i a  Gram  +/v  subterminal o r terminal ; s l i g h t to no swelling o f sporangia  B. b r e v i s  B.  B. licheniformis  Gram  +/v  central. / subterminal / terminal; swelling o f sporangia  Gram  +/v/-  subterminal / terminal; d e f i n i t e swelling o f sporangia  B. s u b t i l u s  Gram + central / paracentral; s l i g h t to no swelling of sporangia  motility  +  +  +  +  +  catalase  +  +  +  V  +  anaerobic  +  +  -  -  -  temperature °C)  maximum - minimum  V.P.  50-55 15 +  r e s i s t a n c e to lysozyme  55-60(65) 15-25  40-60 10-35  65-75 30-45  45-55 5-20  +  -  -  +  -  V  -  V  N a C l 10%  +  -  -  -  +  pH  +  +  V  -  +  5.7  T a b l e 4. -  Continued  Property  Thermophile B. lichenformis  a c i d from - glucose arabinose xylose mannose  B. coagulans  B. b r e v i s  + v v V  B. s t e a r o t h e r m o p h i l u s  B. s u b t i l u s  +  V V V  starch hydrolysis utilization o f c i t r a t e utilization o f propionate NO. '3  NO. ""2  V  ND  ND  V  V  dihydroxyacetone  ND  indole  ND  ND  phenylalanine: deamination  ND  ND  decomposition  ND + v  -  of - tyrosine casein  not done positive f o r 90-100% o f s t r a i n s n e g a t i v e f o r 90-100% o f s t r a i n s c h a r a c t e r inconstant  V  ND  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 & Costilow, 1957; Marshall S Beers, 1967), variation due to different basal media used during testing (Gordon et a l , 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 philic and thermophilic properties (Wolf S Barker, 1968).  are its acidoVirtually all types  grow at 60°C and are capable of initiating growth at pH 5.3. Growth at  81.  low t e m p e r a t u r e s is a f f e c t e d both b y t h e medium a n d n a t u r e o f t h e i n o c u l u m , s p o r e s p r o v i n g more r e s p o n s i v e to lower t e m p e r a t u r e t h a n vegetative cells.  H e n c e it is the maximum t e m p e r a t u r e w h i c h may be o f  g r e a t e s t s i g n i f i c a n c e in c l a s s i f i c a t i o n o f B a c i l l u s t h e r m o p h i l e s (Wolf & Sharp,  1981).  E s s e n t i a l l y , B. coagulans is a f a c u l t a t i v e ; t h e r m o p h i l e ,  g r o w i n g well at 45 - 5 5 ° C . D u r i n g t h e p e r i o d o f i d e n t i f i c a t i o n , it was f o u n d that t h e r m o p h i l i c B a c i l l u s s p e c i e s #1 t h r o u g h #9 d e m o n s t r a t e d a much lower a n t a g o n i s m towards C. olivaceum o n T S Y a g a r .  T h i s v a r i e d from total lack o f  i n h i b i t i o n o f O l i v e g r e e n mold to e x t r e m e l y minimal a n t i b i o t i c c a p a b i l i t y . It seemed that t h e s e c u l t u r e s had o r were u n d e r g o i n g a p o s s i b l e mutation p r o c e s s d u r i n g the c u l t i v a t i o n o n l a b o r a t o r y medium.  F r e s h isolates o f  t h e r m o p h i l e s #1 t h r o u g h #9 w h i c h h a d b e e n s t o r e d at 3°C o n T S Y a g a r s l a n t s were r e g r o w n at 55°C o n f r e s h medium.  T h e s e c u l t u r e s were also  soon s h o w n to h a v e lost t h e i r a n t a g o n i s m a g a i n s t O l i v e g r e e n mold ( u s u a l l y a f t e r a few t r a n s f e r s o n T S Y p l a t e s ) . H o w e v e r , t h e r m o p h i l i c B a c i l l u s #10 maintained a n e x c e l l e n t i n h i b i t o r y effect a g a i n s t C. olivaceum ( F i g u r e 7) s i n c e i t s initial isolation a n d also d u r i n g the identification protocol.  T h u s , o n l y Bacillus coagulans #10  was u s e d f o r a n y f u r t h e r e x p e r i m e n t a t i o n ( h e r e i n a f t e r r e f e r r e d to as Bacillus A O G - " A n t i - O l i v e g r e e n " ) .  F i g u r e 7.  I n h i b i t i o n of O l i v e g r e e n mold b y B a c i l l u s 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 < . 0 1 ) .  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 < . 0 5 ) .  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 • B A C I L L U S ADDED O C H A E T O M I U M ADDED • CHAETOMIUM AND B A C I L L U S 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 B A C I L L U S ADDED B A C I L L U S 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, of sporophores (Nair & Fahy, 1972).  known to stimulate the formation 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% ( /v) w  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 c h o i c e f o r h y d r o p o n i c c u l t i v a t i o n of A.  bisporus.  A s c a n b e seen f r o m F i g u r e 10, w h e n t h e B a c i l l u s was a d d e d to h y d r o p o n i c t r a y s i n 2% Malt e x t r a c t , a r e m a r k a b l e i m p r o v e m e n t i n t h e rate of mycelial development o c c u r r e d (p<.01).  The improved rate of  m y c e l i a l g r o w t h i s s h o w n more v i v i d l y i n t h e r e s p e c t i v e p h o t o g r a p h s o f the h y d r o p o n i c t r a y s .  F i g u r e 11 r e p r e s e n t s a t r a y w i t h o n l y Chaetomium  olivaceum a d d e d - as c a n b e s e e n , r e l a t i v e l y poor s p a w n g r o w t h i s evident;  F i g u r e 12 r e p r e s e n t s t h e e f f e c t o f t h e B a c i l l u s A O G a n d O l i v e  g r e e n mold p r e s e n t at t h e same time.  A s these r e s u l t s show, the mush-  room m y c e l i a f l o u r i s h e d i n t h e p r e s e n c e of t h e B a c i l l u s . M u s h r o o m y i e l d s from t h e 2% Malt e x t r a c t e x p e r i m e n t s ( F i g u r e 13) c o n t a i n i n g O l i v e g r e e n mold o n l y , s h o w e d complete f a i l u r e o f a n y A.  bisporus  f r u i t b o d y formation.  F u r t h e r m o r e , all t r a y s without  B a c i l l u s A O G ( i e . u n f e r m e n t e d 2% Malt e x t r a c t ) s h o w e d v i s i b l e s i g n s o f c o n t a m i n a t i o n b y a n u n i d e n t i f i e d b l u e mold. from t r a y s c o n t a i n i n g  However, the crop yields  t h e B a c i l l u s A O G w e r e maximum - e v e n i n t h e  p r e s e n c e o f t h e C h a e t o m i u m mold. T h e r e s u l t s f o r h y d r o p o n i c c u l t u r e w i t h l i q u i d compost d e m o n s t r a t e d s i g n i f i c a n t biological control d u r i n g both the mycelial g r o w t h  phase  (p<.01) ( F i g u r e 14) a n d d u r i n g f r u i t i n g o f t h e mushroom as t h e y i e l d s d e m o n s t r a t e ( F i g u r e 15) ( a n a l y s i s o f v a r i a n c e p < . 0 5 ) .  T r a y s with only  t h e c o m p e t i t o r C. olivaceum a d d e d d i d not p r o d u c e m u s h r o o m s .  However,  s i m i l a r to t h e 2% Malt e x p e r i m e n t s , t h e maximum y i e l d o c c u r r e d f o r t r a y s c o n t a i n i n g t h e B a c i l l u s A O G a n d C. olivaceum t o g e t h e r . F u r t h e r m o r e ,  88.  2.5  +  • CONTROL  • B A C I L L U S ADDED OCHAETOMIUM ADDED  D B A C I L L U S AND CHAETOMIUM ADDED  2.0 4 -  E u cc  1.5  \LLI  5 LU  i-»  U >-  0.5 -f-  -I  0.0 DAYS Figure 10. Mycelial development in 2% malt extract.  89.  F i g u r e 11.  Poor s p a w n g r o w t h in h y d r o p o n i c t r a y with o n l y O l i v e g r e e n mold p r e s e n t .  . F i g u r e 12.  I m p r o v e d mycelial development d u e to t h e biological protection of Bacillus A O G against  C. olivaceum.  90.  Figure 13. Yield of mushrooms in 2% malt.  91  2.5  -h  o NON-STERILE C O N T R O L • STERILE CONTROL • B A C I L L U S ADDED o CHAETOMIUM AND B A C I L L U S ADDED  2.0.  A CHAETOMIUM ADDED  E u  cn Ui  1.5-U  \~ UJ  <  LLl  U  1.0-r-  >  0.5  0.0  DAYS Figure 14.  Mycelial development in liquid compost.  18.0 -h  j  | CONTROL  [ f f l BACILLUS ADDED H 15.0 4-  BACILLUS AND CHAETOMIUM ADDED  Bjggj CHAETOMIUM ONLY ^  12.04  Q _1 UJ  >-  9.0 -f-  N o n -  6.0  sterile  Sterile  3.0 4-  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 A O G .  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 ( T u r n e r , 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 r u n .  Therefore, it seems the  initial 500ml of liquid compost/tray (ie. water soluble nutrients of  21  18 HOURS OF  F i g u r e 16.  72 FERMENTATION  Fermentation o f 2% malt e x t r a c t b y B a c i l l u s  AOG.  96  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. pure cultures  The successful use of hydroponics coupled with  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 c r o p s .  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'jin 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.  Analysis of Inhibitor Produced b y Bacillus A O G  T h e n e x t s t a g e i n t h i s t h e s i s i n v e s t i g a t i o n was to d e t e r m i n e t h e n a t u r e of t h e i n h i b i t o r p r o d u c e d b y B a c i l l u s A O G a g a i n s t Chaetomium olivaceum (as seen o n T S Y a g a r p l a t e s ) .  A.  E f f e c t o f pH : The f i r s t step i n analysis of the Bacillus A O G  i n h i b i t o r was to d e t e r m i n e i f a s i g n i f i c a n t c h a n g e i n p H was i n v o l v e d o n TSY  media ( i e . s i n c e B. coagulans is a k n o w n a c i d o p h i l e ) .  done b y measuring the pH of T S Y  T h i s was  a g a r p l a t e s at v a r i o u s l o c a t i o n s a f t e r  g r o w t h o f B a c i l l u s A O G ( a n d c h a l l e n g e w i t h C. olivaceum); a n d b y a s e p a r a t e p H g r o w t h s t u d y o f O l i v e g r e e n mold.  The pH of the TSY  agar  p l a t e s was s h o w n to b e r e l a t i v e l y c o n s t a n t t h r o u g h o u t t h e e n t i r e a g a r media ( p H 8.8).  F u r t h e r m o r e , C. olivaceum was o b s e r v e d to b e c a p a b l e  o f g r o w t h t h r o u g h o u t a n e x t e n s i v e p H r a n g e (4-10) a n d t h u s w o u l d not be i n h i b i t e d at p H 8.8.  G o o d g r o w t h at b o t h h i g h a n d low p H l e v e l s b y  O l i v e g r e e n mold has also b e e n r e p o r t e d b y B e a c h (1937).  These data  seem to c l e a r l y c o n c l u d e t h a t a c h a n g e i n p H was not t h e r e s p o n s i b l e e ^ agent f o r inhibition 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 ( 5 5 ° C ) in T S Y 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 A O G . After 6-8d incubation at 55°C in soybean meal. Gram staining revealed the presence of large ovoid spores in Bacillus A O G . with n-butanol.  Extraction of the sterile culture filtrates was carried out This method of extraction (ie. n-butanol) closely resembles  According to Wakman's definition (Stahl, 1969), antibiotics are s u b 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  p r o c e d u r e s commonly e m p l o y e d  producing antibiotics.  f o r i s o l a t i o n o f many B a c i l l u s sp.  H o w e v e r , s i m i l a r to t h e a b o v e e x t r a c t i o n t h e s e  c e l l - f r e e e x t r a c t s also s h o w e d no a c t i v i t y a g a i n s t O l i v e g r e e n mold w h e n tested on T S Y agar. From t h e p o o r r e s u l t s w i t h b r o t h media, i t was d e c i d e d to t r y to e x t r a c t t h e i n h i b i t o r d i r e c t l y from t h e T S Y  agar plates.  T h i s was d o n e  b y r e m o v i n g a s e p t i c a l l y t h e i n h i b i t i o n zone p r o d u c e d b y B a c i l l u s A O G a g a i n s t C. olivaceum (on T S Y a g a r ) a n d s u b s e q u e n t l y a t t e m p t i n g t o leach t h e a n t i b i o t i c o u t of t h e a g a r o n t o f i l t e r p a p e r d i s c s . O n l y t h e e x p e r i m e n t a l t r i a l s u s i n g s t e r i l e a i r to c o n c e n t r a t e t h e e x t r a c t onto t h e s t e r i l e f i l t e r d i s c s was r e a s o n a b l y s u c c e s s f u l ( T a b l e 5 K . O t h e r e x p e r i m e n t s w e r e f o u n d to b e e i t h e r c o n t a m i n a t e d ( i e . d u e to Bacillus A O G spores p r e s e n t ) , or demonstrated no antagonism  towards  C. olivaceum ( p r o b a b l y d u e to low amounts of c o m p o u n d a b s o r b e d onto discs).  T h e s e methods w e r e r e p e a t e d t w i c e w i t h no b e t t e r y i e l d i n g  results.  ii)  Mesophilic extraction: It was s u g g e s t e d t h a t s i n c e p l a t e s s h o w i n g  i n h i b i t i o n o f O l i v e g r e e n mold b y B a c i l l u s A O G h a d b e e n i n c u b a t e d at 25°C, t h e p r o d u c t i o n o f t h i s a n t i b i o t i c c o m p o u n d may o n l y b e c r e a t e d d u r i n g mesophilic growth temperatures.  H e n c e , B a c i l l u s A O G was  i n T S Y b r o t h at 25 a n d 37°C f o r p e r i o d s r a n g i n g from 2-8d.  grown  Following  Table 5.  Extraction of inhibitor from TSY agar discs.  Experiment  Zone of inhibition (mm)  1)  control - uninoculated T S Y 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  ^ v e . of 2 plates  22  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 A O G . It might be postulated that the organism Bacillus AOG only synthesizes 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 ( 5 5 ° C ) but is not required at mesophilic temperatures (25, 37°C) and thus becomes expelled from the cell. AH further extractions of the antibiotic were conducted with culturesat 25°C in T S Y broth for 7d incubation.  Extraction and concentration of  the antibiotic with n-butanol can also be done directly (ie. without freezedrying) 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. Bacillus  AOG.  olivaceum  from cell-free extracts of  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. after storage for 4 months at -15°C no loss in activity was observed.  Even 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. antibiotic thus seems to be hydrophilic in nature.  This  104.  T a b l e 6.  Solvent solubility of antibiotic.  pH  Zone o f I n h 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  pyridine  7.6  3.0  n-butanol  7.0  3.0  isopropanol  7.5  3.0  ethanol  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  6.5  0.0  carbon tetrachloride  6.5  0.0  cyclohexanol  6.5  4.0  Solvent  1  (100%)  petroleum  ether  ^control plates (ie. cyclohexanol same e x t e n t .  o n l y ) also i n h i b i t e d C.  1  olivaceum  to the  105.  E_.  Spectrum of Activity : It was f o u n d t h a t t h e a n t i b i o t i c demon-  s t r a t e d no s u p p r e s s i o n of g r o w t h o f a n y G r a m n e g a t i v e o r g a n i s m s o r of the Gram p o s i t i v e cocci tested.  However, when tested against  7  Bacillus subtilus a n d B. megaterium it was f o u n d to b e q u i t e p o t e n t ( F i g u r e 18).  F u r t h e r m o r e , t h e g r o w t h o f t h e f u n g u s Candida  was also i n h i b i t e d .  lipolytica.  T h e r e f o r e t h i s a n t i b i o t i c c a n b e u s e d e f f e c t i v e l y not  o n l y a g a i n s t Chaetomium olivaceum b u t o t h e r m i c r o o r g a n i s m s as w e l l .  F.  Thin Layer  Chromatography: Because of its h i g h r e s o l v i n g power  a n d s p e e d , t h i n l a y e r c h r o m a t o g r a p h y l e n d s i t s e l f well to t h e s e p a r a t i o n a n d i d e n t i f i c a t i o n o f a n t i b i o t i c s ( S t a h l , 1969).  Therefore, purification  o f t h e a n t i b i o t i c p r o d u c e d b y B a c i l l u s A O G was i n i t i a l l y c a r r i e d o u t b y T L C on Silica Gel plates.  T a b l e 7 shows t h e r e s u l t s o f v a r i o u s s o l v e n t  s y s t e m s u s e d to d e t e r m i n e t h e optimum s e p a r a t i o n o f t h e c r u d e a n t i b i o t i c extract.  T h e p r e s e n c e o f p y r i d i n e i n a n y s o l v e n t s y s t e m was s h o w n to  d e s t r o y t h e b i o l o g i c a l a c t i v i t y o f t h e a n t i b i o t i c a n d h e n c e not f u r t h e r u s e d . It was f o u n d t h a t t h e b e s t r e s u l t s o c c u r r e d w i t h a n n - b u t a n o l - a c e t i c a c i d ( g l a c i a l ) - w a t e r (60:20:20) s o l v e n t s y s t e m followed b y p u r i f i c a t i o n i n a methanol - c h l o r o f o r m - 17% ammonium h y d r o x i d e (40:40:20) T L C s y s t e m . T h e s e s o l v e n t m i x t u r e s a r e two o f t h e most w i d e l y u t i l i z e d s y s t e m s i n s e p a r a t i o n o f a n t i b i o t i c s ( S t a h l , 1969).  106.  a) B. subtilus  cj) B. megaterium F i g u r e 18.  b) C. lipolytica  e) S. lactis  c) S.  f) C .  aureus  lipolytica  Spectrum of activity of Bacillus A O G against various microorganisms.  Table 7.  Solvent systems investigated to determine optimum T L C separation of crude antibiotic.  Observations  Solvent System n-butanol:glacial acetic acid:water (60:20:20)  optimum separation  methanol:chloroform: 17% ammonium hydroxide (40:20:20) fair to good separation  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)  pyridine found to cause loss in biological activity  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) methanol:chloroform (3:97) ethyl acetate:methanol  (87:13)  no movement of sample from origin  108.  A s p r e v i o u s l y mentioned ( M e t h o d s £ M a t e r i a l s ) , f r e e z e - d r i e d extracts o f Bacillus A O G ( i n n-butanol) were streaked across a Silica Gel 60 c h r o m a t o g r a m a n d r u n i n a b u t a n o l - a c e t i c a c i d - w a t e r T L C system.  T o locate t h e b i o l o g i c a l l y a c t i v e b a n d s , a p o r t i o n o f t h e a i r d r i e d  p l a t e was l a y e r e d w i t h T S Y a g a r , s p r a y e d w i t h O l i v e g r e e n mold a n d i n c u b a t e d at 25°C.  Two Ultraviolet light positive (visible under both  short and long wave - however, b r i g h t e r u n d e r s h o r t ) bands with v a l u e s o f .46 a n d .52 ( a n a l y t i c a l 0.2mm plate) r e s u l t e d i n i n h i b i t i o n o f C. olivaceum. TLC  plates.  T h i s i n h i b i t i o n was s h o w n t o b e e x t r e m e l y s t a b l e on t h e T h e l o w e r b a n d ( B a n d I) a p p e a r e d as a b r o a d y e l l o w b a n d  u n d e r s h o r t w a v e U.V. l i g h t ; w h e r e a s t h e u p p e r b a n d ( B a n d II) a p p e a r e d as a n a r r o w l i g h t b l u e b a n d .  F u r t h e r m o r e , B a n d I was o b s e r v e d to  p r o d u c e b u b b l e s w h e n i t was l a y e r e d w i t h T S Y a g a r - t h i s may p o s s i b l y indicate gas p r o d u c t i o n due to t h e possible presence o f perhaps a c a r b o x y l g r o u p o r C 0 g r o u p i n t h e lower T L C 2  band.  B a n d s I a n d II w h e n s p r a y e d w i t h n i n h y d r i n r e a g e n t p r o d u c e d a  :  v i o l e t c o l o r r e a c t i o n i n d i c a t i n g t h e p o s s i b l e p r e s e n c e o f amino a c i d s ( S t a h l , 1969). TLC  P r e s e n c e o f l i p i d s was n o t o b s e r v e d i n e i t h e r b a n d w h e n  c h r o m a t o g r a m s w e r e s p r a y e d w i t h e i t h e r R h o d a m i n e 6G o r alpha-  cyclodextrin.  A phenol-sulphuric acid s p r a y resulted in B a n d T  a p p e a r i n g l i g h t b r o w n a n d B a n d II a medium b r o w n c o l o r a f t e r h e a t i n g p l a t e s f o r 15min at 100°C.  T h e s e c o l o r r e a c t i o n s may d e n o t e t h e p r e s e n c e  o f s u g a r s i n t h e two b i o l o g i c a l l y a c t i v e b a n d s ( S t a h l , 1969). When p r e p a r a t i v e p l a t e s (2.0mm t h i c k n e s s ) w e r e u s e d f o r 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 r u n , 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 h y d r oxide.  Earlier observations showed poor separation had occurred in the  methanol - chloroform - 17% ammonium hydroxide system when using a preparative T L C 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 T S Y agar and challenged with Olive green mold; or sprayed with ninhydrin reagent respectively. Purification of the Bacillus AOG antibiotic by thin layer chromatography was shown to be an extremely long and tedious process (and expensive).  This is especially true when analytical T L C 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  p r e s e n c e o f amino a c i d s i n t h e a n t i b i o t i c (as i n d i c a t e d b y r e a c t i o n on T L C  p l a t e s ) i o n - e x c h a n g e c h r o m a t o g r a p h y was  ninhydrin e m p l o y e d as  the f i r s t c o l u m n method o f s e p a r a t i o n . The  a n t i b i o t i c e x t r a c t ( i n n - b u t a n o l ) was  cation exchange resin ( H  +  c a r e f u l l y layered onto a  form) w i t h a h i g h c r o s s l i n k i n g (X-8)  capacity.  T h i s h i g h c r o s s l i n k i n g made i t e s p e c i a l l y s u i t e d f o r s e p a r a t i o n o f amino a c i d a n d small p e p t i d e c o m p o u n d s ( a c c o r d i n g to m a n u f a c t u r e r ' s s u g g e s t i o n ) . H o w e v e r , as s h o w n b y t h e e l u t i o n p r o f i l e ( F i g u r e 19) o f t h e 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 c o l u m n and was  w a s h e d out d u r i n g  the f i r s t e l u t i o n w i t h d i s t i l l e d w a t e r .  h a v e been due  to  the sample h a v i n g no i o n i c c h a r g e , o r p o s s e s s i n g the same c h a r g e (-)  as  N o n - b i n d i n g may  the f u n c t i o n a l g r o u p o f t h e r e s i n ( i e . S 0  ).  3  In o t h e r w o r d s , d u r i n g  i o n - e x c h a n g e c h r o m a t o g r a p h y , n e u t r a l molecules a n d those h a v i n g  the  same c h a r g e as the f u n c t i o n a l g r o u p ( i e . o f the column) flow t h r o u g h the c o l u m n a n d a r e s e p a r a t e d from any s o r b e d in t h i s c a s e ) .  Or, perhaps non-binding  ions ( i e . t h o s e w i t h a + c h a r g e  may  h a v e been d u e to the  sample b e i n g a much l a r g e r c o m p o u n d t h a n i n i t i a l l y b e l i e v e d .  ii)  Sephadex LH  20: F u r t h e r column chroma-  t o g r a p h y o f t h e a n t i b i o t i c was  c o n d u c t e d w i t h a S e p h a d e x LH  20 t y p e  0  U  8  12  16  20  FRACTION  F i g u r e 19.  21  28  NUMBER  A n t i b i o t i c elution p r o f i l e in cation e x c h a n g e r e s i n .  32  125  112.  resin.  S e p h a d e x LH  20 can be u s e d i n p o l a r s o l v e n t s , and  i s commonly  e m p l o y e d f o r s e p a r a t i o n of b i o l o g i c a l l y a c t i v e s u b s t a n c e s , n a t u r a l s y n t h e t i c p o l y m e r s , and 1967).  and  low m o l e c u l a r w e i g h t s o l u t e s ( J o u s t r a et a l ,  A s well as s e p a r a t i n g c o m p o u n d s a c c o r d i n g  e s s e s b o t h h y d r o p h i l i c and  to s i z e LH  l i p o p h i l i c p r o p e r t i e s , and  20 p o s s ^  can be u s e d f o r  partition chromatography. The  B a c i l l u s AOG  Sephadex LH  e x t r a c t ( i n d i s t i l l e d w a t e r ) was  20 c o l u m n and  l a y e r e d o n t o the  eluted with distilled water.  The  use  of  d i s t i l l e d w a t e r as an e l u a n t 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 l a t e r being freeze-dried for concentration. out t h e U.V. s i m i l a r U.V.  s p e c t r u m and  A l l tubes were scanned t h r o u g h -  the f r a c t i o n s from 5 p e a k s ( d e m o n s t r a t i n g  s p e c t r a ) w,ere pooled and  freeze-dried.  T h i n l a y e r c h r o m a t o g r a p h y 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 b i o l o g i c a l a c t i v i t y ( a g a i n s t C. f r a c t i o n s ( t u b e s 16-20). v i s i b l e u n d e r U.V. to p r e v i o u s T L C  set of pooled  T h e s e two b i o l o g i c a l l y a c t i v e b a n d s w e r e  l i g h t and  results).  olivaceum) from o n l y one  w e r e also n i n h y d r i n p o s i t i v e ( i e . s i m i l a r H o w e v e r , i t seems S e p h a d e x L H  s e p a r a t e t h e two b a n d s ( B a n d I a n d  II)  to a n y  utilizing a single p u r i f i c a t i o n stage with T L C w a t e r ( i e . s i m i l a r R^. v a l u e s ) .  20 d i d not  greater extent than in butanol - acetic a c i d -  S i n c e optimum p u r i f i c a t i o n was  for f u r t h e r a n a l y s i s of the a n t i b i o t i c , it was  necessary  t h e r e f o r e d e c i d e d to c o n t i n u e  w i t h t h i n l a y e r c h r o m a t o g r a p h y as the method of p u r i f i c a t i o n . One was AOG  s h o u l d k e e p i n mind h o w e v e r , that S e p h a d e x L H  20 b y no means  p r o v e n i n e f f e c t i v e as a c h r o m a t o g r a p h y t e c h n i q u e f o r the B a c i l l u s antibiotic.  T h i s r e s i n c o u l d p o t e n t i a l l y be u s e d i n f u t u r e e n d e a v o u r s  113.  at l a r g e s c a l e p u r i f i c a t i o n o f t h e a n t i b i o t i c w i t h p e r h a p s c e r t a i n modifications.  F o r example, experimental  t r i a l s w i t h v a r i a t i o n s i n sample s i z e ,  column size, and t y p e of eluant used might r e s u l t i n better separation o f the b a n d s as c o m p a r e d to t h a t w i t n e s s e d i n t h i 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 S p e c t r u m A n a l y s i s : F i g u r e 20 s h o w s t h e u l t r a v i o l e t spectra of purified Bands I and II. Band I demonstrated a fairly broad peak at 275mu a n d a s m a l l e r peak at a p p r o x i m a t e l y 288mu.  B a n d II was  o b s e r v e d to c o n s i s t o f a s i n g l e peak a t 270mu. T h e a b s o r p t i v i t y c o e f f i c i e n t s w e r e .835 a n d .401 mgmlcm  1  for Bands  I a n d 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 r e p r e s e n t s t h e f l u o r e s c e n t  s p e c t r u m o f T L C B a n d I. A s c a n be s e e n , a s i n g l e peak o c c u r r e d  at 360um -  w i t h a r e l a t i v e i n t e n s i t y o f 42. T h e r e was no f l u o r e s c e n t s p e c t r u m d e t e c t a b l e f o r B a n d I I . T h i s is i n a g r e e m e n t w i t h t h e r e s u l t s o f amino a c i d a n a l y s i s ( S e c t i o n J ) i n w h i c h t r y p t o p h a n was n o t f o u n d i n B a n d II ( i e . t r y p t o p h a n i s t h e major amino acid responsible f o r fluorescence).  F i g u r e 21.  Fluorescent spectrum of T L C  band  I.  116.  J . Amino A c i d A n a l y s i s : A m i n o a c i d a n a l y s i s o f B a n d s I a n d II from T L C chromatography revealed the inhibitor produced by Bacillus to be a p e p t i d e a n t i b i o t i c ( T a b l e 8). in amino a c i d c o m p o s i t i o n .  AOG  Both bands were relatively similar  T h e y b o t h c o n t a i n e d h i g h amounts o f  t y r o s i n e , s e r i n e , aspartic and glutamic acids.  Band I however, contained  a p p r o x i m a t e l y t h r e e times t h e amount o f p h e n y l a l a n i n e as c o m p a r e d to Band II. Furthermore,  B a n d I c o n t a i n e d small amounts o f 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 a n d p r o l i n e - t h e s e amino a c i d s w e r e not d e t e c t e d i n Band II. A l t h o u g h c y s t e i n e was not S H b l o c k e d p r i o r to h y d r o l y s i s ( d u e to small amounts o f c o m p o u n d ) not e v e n a small p e a k was a p p a r e n t f o r t h e p r e s e n c e o f t h i s amino a c i d .  Furthermore,  the breakdown products of  c y s t e i n e ( c y s t e i c a c i d ) from h y d r o l y s i s w i t h p - t o l u e n e s u l f o n i c a c i d w e r e s h o w n not to be p r e s e n t . be a c c o u n t e d  U n u s u a l amino a c i d s o r p e a k s w h i c h c o u l d not  f o r w e r e a l s o not f o u n d .  T h i s amino a c i d p r o f i l e also i n d i c a t e s t h e a n 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 f e a t u r e m i g h t be r e s p o n s i b l e f o r t h e e a r l i e r  o b s e r v a t i o n s ( S e c t i o n C) a s s o c i a t e d w i t h i o n - e x c h a n g e c h r o m a t o g r a p h y ( i e . n o n - b i n d i n g to r e s i n ) . T h e s e r e s u l t s pose many i n t e r e s t i n g q u e s t i o n s r e g a r d i n g t h e p e p t i d e antibiotics produced by Bacillus AOG. organism  F o r example, why does the  p r o d u c e 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 , w h y a r e t h e y  s i z e d ( e s p e c i a l l y at o n l y m e s o p h i l i c t e m p e r a t u r e s ) , a n d a r e t h e s e  synthe-  Table 8 .  Amino acid analysis of Bands I and Band I (g/IOOg amino acid)  II.  Band II (g/IOOg amino acid)  Aspartic acid  19.90  24.40  Phenylalanine  18.90  5.66  Tyrosine  14.51  17.63  Serine  14.19  17.11  Glutamic acid  12.34  17.06  Threonine  5.72  8.47  Leucine  2.80  1.72  Tryptophane  2.80  0.00  Proline  1.52  0.00  Isoleucine  0.60  0.98  Valine  0.54  0.66  Alanine  0.41  0. 33  Glycine  0.40  0.98  Lysine  0.22  0.00  Histidine  0.12  0.00  94.97  95.00  Total  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 a l , 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). Bacillus subtilus  For example.  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). T h e  antibiotics of the Bacillaceae are similar in the following general p r o p 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 m R N A o r r i b o s o m e s as i n t h e case o f normal p r o t e i n s y n t h e s i s , and  4) p e p t i d e 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 i m p o r t a n t c e l l u l a r p r o c e s s e s ( e g . DNA  synthesis).  L i t t l e p r o g r e s s has been made i n e l u c i d a t i n g t h e f u n c t i o n ( s ) of a n t i b i o t i c s i n the p r o d u c i n g o r g a n i s m ( B u ' L o c k , 1961).  Generally  members of t h e B a c i l l a c e a e a r e c h a r a c t e r i z e d b y t h e i r a b i l i t y to p r o d u c e r e s i s t a n t e n d o s p o r e s and ( H o d g s o n , 1970).  i n t h i s c o u r s e , to e l a b o r a t e p e p t i d e a n t i b i o t i c s  It t h u s seems a p p r o p r i a t e to assume t h a t t h e 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 i n t h e c e l l s f r o m which they have been e x c r e t e d .  A s 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 i s a means of k e e p i n g t h e c e l l u l a r m a c h i n e r y i n w o r k i n g d u r i n g the time w h e n c e l l g r o w t h is not p o s s i b l e d u e  to  order  unfavorable  conditions. H o d g s o n (1970) b e l i e v e s t h a t p e p t i d e a n t i b i o t i c s m i g h t be u s e d i n s e v e r a l w a y s b y an o r g a n i s m s u c h as m o d i f i e r s o f t h e cell membrane; f o r e x a m p l e , ion c a r r i e r s , m o d i f y i n g  permeability characteristics.  Furthermore,  S a d o f f (1972) s t a t e s t h a t t h e p e p t i d e a n t i b i o t i c s p r o d u c e d b y the b a c i l l i e x h i b i t e f f e c t s u p o n membrane s y n t h e s i s and " f u n c t i o n , cell wall s y n t h e s i s , and  nucleic acid synthesis.  It:was~contended t h a t t h e a n t i b i o t i c w h e n  p r o d u c e d b y an o r g a n i s m , may  act as s e l e c t i v e m o d i f i e r s of c e l l f u n c t i o n -  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 . q u a n t i t i e s r e q u i r e d i n s p o r u l a t i o n m i g h t amount to r e l a t i v e l y few per cell.  Thus antibiotic production  by B a c i l l u s AOG  probably  The  molecules represents  an a m p l i f i c a t i o n of a normal e v e n t w h i c h o c c u r s to a l e s s e r d e g r e e i n a l l  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. active cellular growth of B. licheniformis  For example, during  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 ( 2 5 ° 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.  A n abundance of amino acids at  mesophilic temperatures might normally result in cell death due to u n 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 r e pression or is under growth rate control; manipulations which affect those controls (eg. possibly large fluctuations in temperature) would be expected  121.  to m o d i f y t h e temporal r e l a t i o n s h i p b e t w e e n a n t i b i o t i c s y n t h e s i s a n d growth. S t i l l u n d e r c u r r e n t c o n s i d e r a t i o n , is the possibility that antibiotics f u n c t i o n to k i l l o r i n h i b i t t h e g r o w t h o f o t h e r o r g a n i s m s i n n a t u r e , t h e r e b y p r o v i d i n g a c o m p e t i t i v e a d v a n t a g e to t h e p r o d u c i n g ( G o t t l i e b , 1976).  One  organism  m i g h t s p e c u l a t e t h a t B a c i l l u s AOG  has e v o l v e d to  p r o d u c e a n t i b i o t i c s at o n l y m e s o p h i l i c temperatures' b e c a u s e t h e r e is a m u c h l a r g e r n u m b e r o f m e s o p h i l i c c o m p e t i t i v e o r g a n i s m s as c o m p a r e d to t h o s e at t h e r m o p h i l i c t e m p e r a t u r e s - e s p e c i a l l y i n mushroom compost from w h e r e B a c i l l u s AOG The  was  initially isolated.  t h r e e main c l a s s e s o f a n t i f u n g a l p e p t i d e s ( p o s s e s s i n g o n l y s l i g h t  antibacterial a c t i v i t y ) produced by Bacillus sp. are the bacillomycins, m y c o b a c i l l i n s , a n d 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). Bacillus AOG  S i m i l a r to t h e  i n h i b i t o r , t h e y h a v e p r o v e n to be e x t r a c e l l u l a r , a c i d i c  ( f u n g i s t a t i n - b a s i c ) , c o n t a i n no s u l p h u r amino a c i d s , a n d a r e heatstable peptides.  T h e y are also soluble in polar, and insoluble in non-polar  o r g a n i c s o l v e n t s ( S h a r o n et a l , 1954).. Most o f t h e s e a n t i b i o t i c s , a l t h o u g h i s o l a t e d m a n y - y e a r s ago a r e k n o w n o n l y i n t h e i r q u a l i t a t i v e E x c e p t f o r f u n g i s t a t i n "(which c o n t a i n s u n i d e n t i f i e d  composition.  amino a c i d s ) , b o t h  the  b a c i l l o m y c i n a n d m y c o b a c i l l i n f a m i l y c o n t a i n o n l y k n o w n amino a c i d s w i t h no o t h e r components.However, closer examination  reveals that severe d i s p a r i t i e s o c c u r  between these Bacillus antibiotic classes and the Bacillus AOG a n d t h u s can not be g r o u p e d i n t o t h e s e f a m i l i e s .  compound -  For example, both  122.  mycobacillin and bacillomycin are n i n h y d r i n negative and  ( S h a r o n et a l , 1954),  s i g n i f i c a n t d i f f e r e n c e s i n c o n t e n t o f s p e c i f i c amino a c i d s ( e g . b a c i l l o -  m y c i n c o n t a i n s o n l y 5 amino a c i d s ) , U.V. solubility  adsorption, and solvent  occurs.  A f u r t h e r extensive examination of the literature c o n c e r n i n g fungal p e p t i d e a n t i b i o t i c s r e v e a l s as o f y e t , no k n o w n a n t i b i o t i c s w i t h i d e n t i c a l p r o p e r t i e s ( e g . amino a c i d s , U.V.  s p e c t r a , s o l v e n t s o l u b i l i t y etc.) as  that o b s e r v e d i n this thesis investigation  of the Bacillus AOG  antibiotic.  O n e may r e a s o n a b l y c o n c l u d e t h e r e f o r e , t h a t t h e i n h i b i t o r p r o d u c e d b y Bacillus AOG  i s a u n i q u e a n t i b i o t i c c o m p o u n d a n d t h u s has b e e n g i v e n  t h e name C h a e t o m a c i n .  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).  T h e 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.  T h e 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 T S Y 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 thermophiles 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.  a n d i s v e r y l i g h t to f a c i l i t a t e t r a n s p o r t a t i o n ) - p o s s i b l y l e a d i n g to eventual continuous culture techniques. Bacillus AOG  was s h o w n to p r o d u c e a p o t e n t a n t i b i o t i c (named  C h a e t o m a c i n ) at m e s o p h i l i c t e m p e r a t u r e s .  Extraction of this compound  w i t h n - b u t a n o l r e v e a l e d i t to be a s t a b l e s u b s t a n c e , e f f e c t i v e a g a i n s t other fungi and Bacillus species.  Purification through thin layer chroma-  t o g r a p h y r e v e a l e d two c o m p o u n d s w i t h c l o s e R^ v a l u e s . A m i n o a c i d a n a l y s i s s h o w e d t h e s e two b a n d s to be s i m i l a r i n s t r u c t u r a l  compostion.  E x a m i n a t i o n o f t h e l i t e r a t u r e r e v e a l s no o t h e r p r e v i o u s l y i s o l a t e d a n t i b i o t i c s w h i c h a r e i d e n t i c a l to t h e i n h i b i t o r s f o u n d i n t h i s t h e s i s study.  Chaetomacin  c o u l d p o t e n t i a l l y be e x t e n d e d i n t o t h e p r e p a r a t i o n  of d i f f e r e n t i a l media a n d i n t h e p r o t e c t i o n o f foods a n d p l a n t s from fungal invasion.  126.  LITERATURE  CITED  1.  A l l e n , M . B . 1953.  T h e thermophilic aerobic sporeforming bacteria.  2.  A n o n . 1981.  3.  A t k i n s , F . C . 1972. Mushroom growing today.  4.  Aulin-Erdtman, G . 1949.  5.  Baker, R. 1968. 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Micb. 95:313-323.  APPENDICES  APPENDIX  A  Statistical analysis of mycelial diameters.  CONVENTIONAL  LINE  1 : NOTH DAY  LINE 2  INDIVIDUAL  1 2  SIMREG  FOR  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  B= 0.18393E+00 B= 0.27143E+00  SEY=0.6594E-01 SEY=0.1038E+00  R= R=  0.9887 0.9872  DF= DF=  6. 6.  R=  0.9351  DF=  13.  B= 0.22768E+00  SEY=0.1864E+00  LINES  A = 0.34857E+00 A= 0.25714E+00 SIMREG  3  : BAC DAY  POOLED DATA  A= 0.30286E+00  COVARIANCE  ANALYSIS  TEST FOR HOMOGENEITY OF RESIDUAL F,  5./  5.=  TEST  FOR  SLOPES  F,  1/  TEST FOR F,  1/  OVERALL F,  2./  10.=  0.403E+00  0.142E+02  LEVELS 11.=  0.141E+02  TEST 10.=  0.226E+02  VARIANCES  DEGREES OF FREEDOM  NOTH DAY  LINE 2  INDIVIDUAL  : B+C DAY  A= 0.34857E+00 A= O.eOOOOE-01 SIMREG  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  B= 0.18393E+00 B= 0.28071E+00  SEY=0.6594E-01 SEY=0.1104E+O0  R= R=  0.9887 0.9865  DF= DF=  6. S.  B= 0.23232E+00  SEY=0.1437E+00  R=  0.9614  DF=  13.  SIMREG L I N E S  FOR POOLED  DATA  A= O.20429E+0O  COVARIANCE  ANALYSIS  TEST  FOR HOMOGENEITY OF RESIDUAL VARIANCES  F,  5./  5.=  0.357E+00  TEST FOR SLOPES F. TEST F,  1/ FOR 1/  OVERALL F,  2./  DEGREES OF FREEDOM  10.=  O.159E+02  LEVELS 11.= 0.175E+01 TEST 10.= 0.999E+01  CONVENTIONAL  LINE  1 : NOTH DAY  1 2  LINE 2  : CHAE DAY  INDIVIDUAL  SIMREG  A= 0.34857E+00 A= 0.45714E+00 SIMREG  3  FOR  CORRELATION COEFFICIENT  B= 0.18393E+00 B= 0.14286E+00  SEY=0.G594E-01 SEY=0.6761E-01  R= R=  0.9887 0.9806  DF= DF=  6. 6.  R=  0.9746  DF=  13.  B= 0.16339E+00  SEY=0.8118E-01  POOLED DATA  A= 0.40286E+00  COVARIANCE  ANALYSIS  T E S T FOR HOMOGENEITY OF RESIDUAL F.  5./  5.=  TEST  FOR  SLOPES  F. TEST F,  1/ FOR 1/  OVERALL F,  2./  DEGREES OF FREEDOM  STANDARD ERROR OF ESTIMATE  LINES  10.=  0.951E+00  0.530E+01  LEVELS 11.=  0.175E+01  TEST 10.=  0.387E+01  VARIANCES  CONVENTIONAL  LINE 1  BAC DAY  LINE  B+C DAY  2  INDIVIDUAL  SIMREG  LINES  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  DEGREES OF FREEDOM  B= B=  0.27143E+00 0.28071E+OO  SEY=0.1038E+O0 SEY=0.1104E+00  R= R=  0.9872 0.9865  DF= DF=  6. 6.  SIMREG FOR POOLED DATA A= 0.15857E+00 B=  O.27607E+0O  SEY=0.1309E+00  R=  0.9767  DF =  13.  A= 0.25714E+00 A= O.GOCOOE-01  COVARIANCE  ANALYSIS  TEST  FOR HOMOGENEITY OF RESIDUAL  F,  5./  5.=  TEST  FOR  SLOPES  F, TEST F,  1/ FOR 1/  OVERALL F,  2./  10.=  0.884E+00  0.105E+00  LEVELS 11.=  0.849E+01  TEST 10.=  0.395E+01  VARIANCES  CONVENTIONAL  LINE  1  2  CHAE DAY  INDIVIDUAL  SIMREG  BAC DAY  LINE  LINES  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  DEGREES OF FREEDOM  B= B=  0.27143E+00 0.14286E+00  SEY=0.1038E+00 SEY=0.6761E-01  R= R=  0.9872 0.9806  DF= DF=  6. 6.  SIMREG FOR POOLED DATA A= O.35714E+O0 B=  0.20714E+O0  SEY=0.2334E+0O  R=  0.8866  DF=  13.  A= A=  0.25714E+00 0.45714E+00  COVARIANCE  ANALYSIS  TEST  FOR HOMOGENEITY OF RESIDUAL  F,  5./  5.=  TEST  FOR  SLOPES  F, TEST F,  1/ FOR 1/  OVERALL F,  2./  10.=  0.236E+01  0.302E+02  LEVELS 11.=  0.123E+02  TEST 10.=  0.376E+02  VARIANCES  CONVENTIONAL  LINE 1  2  CHAE DAY  INDIVIDUAL  SIMREG  B+C DAY  LINE  LINES  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  DEGREES FREEDOM  B= B=  0.28071E+00 O.14286E+00  SEY=0.1104E+00 SEY=0.6761E-01  R= R=  0.9865 0.9806  DF= DF=  6. 6.  SIMREG FOR POOLED DATA A= 0.25857E+00 B=  0.21179E+00  SEY=0.190OE+0O  R=  0.9235  DF=  13.  A= A=  0.6CO00E-01 0.45714E+00  COVARIANCE  ANALYSIS  T E S T FOR HOMOGENEITY OF RESIDUAL F.  5./  TEST FOR F,  1/  T E S T FOR F,  1/  OVERALL F,  THERE ARE  (F3.0.4F6.2)  2./  5 VARIABLES AND  5.=  0.267E+01  SLOPES 10.=  0.318E+02  LEVELS 11.=  0.262E+01  TEST 10.=  0.208E+02  6 PAIRS OF  LINES  VARIANCES  CONVENTIONAL  LINE 1  NOTH DAY  BAC DAY  LINE 2  INDIVIDUAL  SIMREG  1 2  A= 0.34857E+00 A= 0.25714E+OQ  3  SIMREG FOR POOLED A= 0.30286E+00  LINES  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  B= B=  0.18393E+00 0.27143E+QO  SEY=0.G594E-01 SEY=0.1038E+O0  R= R=  0.9887 0.9872  DF= DF=  6. 6.  B=  0.22768E+00  SEY=0.1864E+O0  R=  0.9351  DF=  13.  DATA  COVARIANCE  ANALYSIS  TEST  FOR HOMOGENEITY OF RESIDUAL VARIANCES  F,  5./  5.=  TEST  FOR  SLOPES  F, TEST F,  1/ FOR 1/  OVERALL F,  2./  DEGREES OF FREEDOM  10.=  0.403E+00  0.142E+02  LEVELS 11.=  O.141E+02  TEST 10.=  0.226E+02  CONVENTIONAL  LINE  1 : NOTH DAY  LINE 2  INDIVIDUAL  1 2  SIMREG  FOR POOLED  CORRELATION COEFFICIENT  B= O.18393E+00 B= 0.28071E+OO  SEY=0.6594E-01 SEY=0.1104E+00  R= R=  0.9887 0.9865  DF= DF=  6. 6.  R=  0.9614  DF=  13.  B= 0.23232E+0O  SEY=0.1437E+00  DATA  A= 0.20429E+00  COVARIANCE  ANALYSIS  T E S T FOR HOMOGENEITY OF RESIDUAL VARIANCES F.  5./  TEST  FOR SLOPES  F. TEST F,  1/ FOR 1/  OVERALL F.  2.1  DEGREES OF FREEDOM  STANDARD ERROR OF ESTIMATE  LINES  A= 0.34857E+00 A= 0.60OOOE-01 SIMREG  3  : B+C DAY  5.=  10.=  0.357E+CO  0.159E+02  LEVELS 1 1 .= 0.175E+01 TEST 10.= 0.999E+01  CONVENTIONAL  LINE  1 : NOTH DAY  LINE  2  CHAE DAY  INDIVIDUAL  SIMREG  LINES  A= 0.34857E+00 A= 0.45714E+00 SIMREG FOR POOLED A= 0.40286E+00  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  B= B=  O.18393E+00 O.14286E+OQ  SEY=0.6594E-01 SEY=0.6761E-01  R= R=  0.9887 0.9806  DF= DF=  6. 6.  B=  0.16339E+00  SEY=0.8118E-01  R=  0.9746  DF=  13.  DATA  COVARIANCE  ANALYSIS  TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F,  5./  5.=  0.951E+OO  TEST FOR SLOPES F, TEST F,  1/ FOR 1/  OVERALL F,  2./  DEGREES OF FREEDOM  10.= 0.530E+01 LEVELS 11.=  O.175E+01  TEST 10.= 0.387E+01  CONVENTIONAL  LINE 1  BAC DAY  LINE  2  INDIVIDUAL  A= A=  B+C DAY SIMREG  LINES  B= 0.27143E+00 B= O.28O71E+0O  0.25714E+00 0.6000OE-01  SIMREG FOR POOLED DATA A= 0.15857E+O0 B=  0.27607E+00  COVARIANCE  5./  TEST FOR F,  1/  TEST FOR F,  1/  OVERALL F.  2./  5.=  0.884E+00  SLOPES 10.=  O.105E+00  LEVELS 11.=  0.849E+01  TEST 10.=  CORRELATION COEFFICIENT  SEY=0.1038E+00 SEY=0.1104E+00  R= R=  0.9872 0.9865  DF= DF=  G. 6.  SEY=0.1309E+OQ  R=  0.9767  DF =  13.  ANALYSIS  TEST FOR HOMOGENEITY OF RESIDUAL F,  STANDARD ERROR OF ESTIMATE  0.395E+01  VARIANCES  DEGREES OF FREEDOM  CONVENTIONAL  LINE 1  BAC DAY  LINE  2  CHAE DAY  INDIVIDUAL SIMREG  LINES  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  DEGREES FREEDOM  B= B=  0.27143E+00 O.14286E+00  SEY=0.1038E+00 SEY=0.6761E-01  R= R=  0.9872 0.9806  DF= DF=  6. 6.  SIMREG FOR POOLED DATA A= 0.35714E+00 B=  0.20714E+00  SEY=0.2334E+0O  R=  0.8866  DF=  13.  A= A=  0.25714E+00 0.45714E+00  COVARIANCE  ANALYSIS  TEST FOR HOMOGENEITY OF RESIDUAL F,  5./  5.=  TEST  FOR  SLOPES  F, TEST F,  1/ FOR 1/  OVERALL F.  2./  10.=  0.236E+01  0.302E+02  LEVELS 11.=  0.123E+02  TEST 10.=  0.37SE+02  VARIANCES  CONVENTIONAL  LINE  1 : B+C DAY  LINE  2  : CHAE DAY  INDIVIDUAL SIMREG  1 2  LINES  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  DEGREES OF FREEDOM  B= B=  0.28071E+OO O.14286E+0O  SEY=0.1104E+00 SEY=0.6761E-01  R= R=  0.9865 0.9806  DF= DF=  6. 6.  SIMREG FOR POOLED DATA A= 0.25857E+O0 B=  0.21179E+00  SEY=0.1900E+00  R=  0.9235  DF=  13.  A= 0.60000E-01 A= 0.45714E+O0  COVARIANCE  ANALYSIS  T E S T FOR HOMOGENEITY OF RESIDUAL F,  5./  TEST FOR F.  1/  TEST FOR F.  1/  OVERALL F,  2./  5.=  0.267E+01  SLOPES 10.=  0.318E+02  LEVELS 11.=  0.262E+01  TEST 10.=  0.208E+02  VARIANCES  TWO  PERCENT  LINE 1  MALT  NOTH DAY  LINE  2  INDIVIDUAL  BAC DAY SIMREG  LINES  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  DEGREES FREEDOM  B= B=  0.23250E+00 0.24536E+00  SEY=0.1052E+00 SEY=0.1008E+00  R= R=  0.9822 0.9853  DF= DF=  6. 6.  SIMREG FOR POOLED DATA A= 0.17214E+00 B=  0.23893E+00  SEY=0.1259E+00  R=  0.9715  DF=  13.  A= 0.12143E+00 A = 0.22286E+00  COVARIANCE  ANALYSIS  T E S T FOR HOMOGENEITY OF RESIDUAL F,  5./  5.=  TEST  FOR  SLOPES  F, TEST F.  1/ FOR 1/  OVERALL F,  2./  10.=  O.109E+01  0.218E+0O  LEVELS 11.=  0.830E+01  TEST 10.=  0.396E+01  VARIANCES  OF  TWO  LINE  PERCENT  MALT  1 : NOTH DAY  LINE 2  : B+C DAY STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  B= 0.23250E+00 B= 0.21643E+00  SEY=0.1052E+00 SEY=0.8924E-01  R= R=  0.9822 0.9852  DF= DF=  6. 6.  R=  0.9789  DF=  13.  B= 0.22446E+00  SEY=0.1011E+OO  INDIVIDUAL SIMREG L I N E S  1 2  A= 0.12143E+00 A= 0.26857E+00 SIMREG  3  FOR POOLED  DATA  A= 0.1950OE+0O  COVARIANCE  ANALYSIS  TEST  FOR HOMOGENEITY OF RESIDUAL VARIANCES  F,  5./  TEST  FOR SLOPES  F,  1/  TEST FOR F,  1/  OVERALL F,  2./  5.=  10.=  0.139E+01  0.380E+O0  LEVELS 11.= 0.268E+01 TEST 10.= 0.145E+01  DEGREES OF FREEDOM  TWO  LINE  PERCENT  MALT  1 : NOTH DAY  1 2  LINE 2  : CHAE DAY  INDIVIDUAL  SIMREG  A= 0.12143E+00 A= 0.36429E+00 SIMREG  3  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  B= 0.23250E+0O B= 0.10714E+0O  SEY=0.1052E+00 SEY=0.4629E-01  R= R=  B= 0.16982E+00  SEY=0.2082E+00  LINES  DEGREES OF FREEDOM  0.9822 0.9837  DF= DF=  6. 6.  0.8697  DF=  13.  FOR POOLED DATA  A = 0.24286E+00  COVARIANCE  ANALYSIS  TEST  FOR HOMOGENEITY OF RESIDUAL VARIANCES  F,  5./  5.=  TEST  FOR  SLOPES  F,  1/  TEST FOR F,  1/  OVERALL F,  2./  10.=  0.516E+01  0.333E+02  LEVELS 11.= 0.900E+01 TEST 10.=  0.344E+02  TWO  PERCENT  LINE 1  MALT  BAC DAY  LINE  2  INDIVIDUAL  1 2  A= A=  : B+C DAY SIMREG  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  0.24536E+CO 0.21643E+00  SEY=0.1008E+00 SEY=0.8924E-01  R= R=  0.9853 0.9852  DF= DF=  6. 6.  B = O.23089E+0O  SEY=0.9977E-01  R=  0.9806  DF=  13.  LINES  0.22286E+00 0.26857E+00  B= B=  SIMREG FOR POOLED A= 0.24571E+OO  DATA  COVARIANCE  ANALYSIS  TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F,  5./  TEST  FOR SLOPES  F,  1/  TEST FOR F,  1/  OVERALL F,  2./  DEGREES OF FREEDOM  5.=  0.128E+01  10.= 0.129E+01 LEVELS 11.= 0.184E+01 TEST 10.= 0.159E+01  TWO  LINE  PERCENT  MALT  1 : BAC DAY  LINE 2  CHAE DAY  INDIVIDUAL SIMREG  1 2  LINES  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  DEGREES FREEDOM  B= B=  0.24536E+00 0.10714E+0O  SEY=0.1008E+00 SEY=0.4629E-01  R= R=  0.9853 0.9837  DF= DF=  6. 6.  SIMREG FOR POOLED DATA A= 0.29357E+00 B=  0.17625E+O0  SEY=0.2771E+00  R=  0.8085  DF=  13.  A= A=  0.2228GE+00 O.36429E+0O  COVARIANCE  ANALYSIS  T E S T FOR HOMOGENEITY OF RESIDUAL F,  5./  5.=  TEST  FOR  SLOPES  F, TEST F,  1/ FOR 1/  OVERALL F,  2./  10.=  OF  VARIANCES  0.474E+01  0.435E+02  LEVELS 11.=  O.198E+02  TEST 10.=  0.699E+02  o  TWO  PERCENT  LINE 1  MALT  B+C DAY  LINE  2  CHAE DAY  INDIVIDUAL SIMREG  LINES  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  DEGREES FREEDOM  B= B=  0.21643E+O0 0.10714E+00  SEY=0.8924E-01 SEY=0.4629E-01  R= R=  0.9852 0.9837  DF= DF=  6. 6.  SIMREG FOR POOLED DATA A = 0.31643E+00 B=  O.16179E+00  SEY=0.2284E+00  R=  0.8371  DF=  13.  A= A=  0.26857E+O0 0.36429E+00  COVARIANCE  ANALYSIS  TEST  FOR HOMOGENEITY OF RESIDUAL  F,  5./  5.=  TEST  FOR  SLOPES  F, TEST F,  1/ FOR 1/  OVERALL F,  2./  10.=  0.372E+01  0.331E+02  LEVELS 11.=  0.206E+02  TEST 10.=  0.569E+02  VARIANCES  OF  L I Q U I D COMPOST  LINE  1 : NOTH DAY  LINE  2  : BAC DAY STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  0.16679E+00 0.23G07E+00  SEY=0.9208E-01 SEY=0.7203E-01  R= R=  0.9738 0.9918  DF= DF=  6. 6.  SIMREG FOR POOLED DATA A= 0.34286E+00 B= 0.20143E+00  SEY=0.1102E+00  R=  0.9694  DF=  13.  INDIVIDUAL SIMREG  A= A=  LINES  B= B=  0.45429E+00 0.23143E+00  COVARIANCE  ANALYSIS  TESt  FOR HOMOGENEITY OF RESIDUAL VARIANCES  F.  5./  TEST FOR F,  1/  TEST FOR F,  1/  OVERALL F.  2./  5.=  DEGREES OF FREEDOM  0.163E+01  SLOPES 10.= 0.983E+01 LEVELS 11.=  0.837E+00  TEST 10.=  0.5G7E+01  ro  LIQUID COMPOST  LINE 1  NOTH DAY  LINE 2  B+F DAY  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  B= O.16679E+00 B= 0.24679E+00  SEY=0.9208E-01 SEY=O.S545E-01  R= R=  0.9738 0.9895  DF= DF=  6. 6.  SIMREG FOR POOLED DATA A = 0.28857E+00 B= 0.20679E+00  SEY=0.1187E+00  R=  0.9665  DF=  13.  INDIVIDUAL SIMREG LINES A= 0.45429E+00 A= 0.12286E+00  DEGREES OF FREEDOM  COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL F,  5./  5.=  VARIANCES  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  co  L I Q U I D COMPOST  LINE  1 : NOTH DAY  LINE  2  INDIVIDUAL  1 2  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  B= O.16G79E+00 B = O.15929E+0O  SEY=0.9208E-01 SEY=0.4890E-01  R= R=  0.9738 0.9917  DF= DF=  6. 6.  B= 0.16304E+OO  SEY=0.9594E-01  R=  0.9648  DF=  13.  SIMREG L I N E S  A= 0.45429E+00 A= O.35857E+0O SIMREG  3  : B+C DAY  FOR POOLED  DATA  A= 0.40643E+00  COVARIANCE  ANALYSIS  T E S T FOR HOMOGENEITY OF RESIDUAL VARIANCES F,  5./  T E S T FOR F,  1/  TEST FOR F,  1/  OVERALL F,  2./  DEGREES OF FREEDOM  5.=  0.355E+01  SLOPES 10.=  O.145E+00  LEVELS 11.=  0.110E+02  TEST 10.= 0.516E+01  L I Q U I D COMPOST  LINE  1 : NOTH DAY  LINE  2  : CHAE DAY  DEGREES OF FREEDOM  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  B= 0.16679E+00 B= 0.48214E-01  SEY=0.9208E-01 SEY=0.2401E-01  R= R=  0.9738 0.9786  DF= DF=  6. 6.  SIMREG FOR POOLED DATA A= 0.51357E+00 B= 0. 10750E+00  SEY=0.2389E+00  R=  0,6970  DF=  13.  INDIVIDUAL SIMREG  A= A=  LINES  0.45429E+00 0.57286E+00  COVARIANCE  ANALYSIS  T E S T FOR HOMOGENEITY OF RESIDUAL F.  5./  5.=  TEST  FOR  SLOPES  F, TEST F.  1/ FOR 1/  OVERALL F,  2./  10.=  0.147E+02  0.435E+02  LEVELS 11.=  0.201E+02  TEST 10.=  0.706E+02  VARIANCES  L I Q U I D COMPOST  LINE 1  BAC DAY  LINE 2  INDIVIDUAL  1 2  A= A=  : B+F DAY SIMREG  LINES  0.23143E+00 O.12286E+00  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  DEGREES OF FREEDOM  B= B=  0.23607E+00 0.24679E+00  SEY=O.7203E-O1 SEY=0.8545E-01  R= R=  0.9918 0.9895  DF= DF=  6. 6.  SIMREG FOR POOLED DATA A = 0.17714E+00 B=  0.24143E+0O  SEY=0.8123E-01  R=  0.9881  DF=  13.  COVARIANCE  ANALYSIS  T E S T FOR HOMOGENEITY OF RESIDUAL VARIANCES F,  5./  T E S T FOR F, TEST F,  1/ FOR 1/  OVERALL F.  2./  5.=  0.710E+00  SLOPES 10.=  0.257E+00  LEVELS 11.= 0.260E+01 TEST 10.= 0.134E+01  L I Q U I D COMPOST  LINE 1  BAC DAY  LINE  2  INDIVIDUAL  B+C DAY SIMREG  LINES  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  DEGREES FREEDOM  B= B=  0.23607E+00 O.15929E+00  SEY=0.7203E-01 SEY=0.4890E-O1  R= R=  0.9918 0.9917  DF= DF=  6. 6.  SIMREG FOR POOLED DATA A= O.295O0E+0O B=  0.19768E+00  SEY=0.1396E+CO  R=  0.9505  DF=  13.  A= A=  0.23143E+00 O.35857E+0O  COVARIANCE  ANALYSIS  TEST  FOR HOMOGENEITY OF RESIDUAL  F,  5./  5.=  TEST  FOR  SLOPES  F, TEST F,  1/ FOR 1/  OVERALL F,  2./  10.=  0.217E+01  0.218E+02  LEVELS 11.=  0.104E+02  TEST 10.=  0.259E+02  VARIANCES  L I Q U I D COMPOST  LINE  1 : BAC DAY  A= A=  LINE  2  : CHAE DAY  INDIVIDUAL  SIMREG  LINES  B= 0.23607E+00 B= 0.48214E-01  0.23143E+00 0.57286E+00  SIMREG FOR POOLED DATA A= 0.40214E+00 B=  0.14214E+00  COVARIANCE  5./  T E S T FOR F,  1/  T E S T FOR F,  1/  OVERALL F.  1.1  5.=  CORRELATION COEFFICIENT  SEY=0.7203E-01 SEY=0.2401E-01  R= R=  0.9918 0.9786  DF= DF=  6. 6.  SEY=0.3043E+00  R=  0.7103  DF*  13.  ANALYSIS  TEST FOR HOMOGENEITY OF RESIDUAL F,  DEGREES OF FREEDOM  STANDARD ERROR OF ESTIMATE  VARIANCES  0.900E+01  SLOPES 10.=  O.171E+03  LEVELS 11.=  O.124E+02  TEST 10.=  0.188E+03  ov CO  L I Q U I D COMPOST  LINE  1 : B+F DAY  LINE  2  B+C DAY  INDIVIDUAL SIMREG  A= A=  LINES  O.12286E+00 0.35857E+00  B = 0.24679E+00 B= 0.15929E+00  SIMREG FOR POOLED DATA A= 0.24071E+OO B=  0.20304E+00  COVARIANCE  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  DEGREES OF FREEDOM  SEY=0.8545E-01 SEY=0.4890E-01  R= R=  0.9895 0.9917  DF= DF=  6. 6.  SEY=0.1295E+O0  R=  0.9590  DF=  13.  ANALYSIS  T E S T FOR HOMOGENEITY OF RESIDUAL VARIANCES F,  5./  5.=  0.305E+01  TEST FOR SLOPES F,  1/  TEST FOR F,  1/  OVERALL F.  2./  10.=  0.221E+02  LEVELS 11.= 0.323E+01 TEST 10.=  0.158E+02  (£>  L I Q U I D COMPOST  LINE 1  1 2  B+F DAY  A= A=  LINE  2  : CHAE DAY  INDIVIDUAL  SIMREG  LINES  0.12286E+00 0.57286E+00  B= 0.24679E+00 B= 0.48214E-01  SIMREG FOR POOLED DATA A= 0.34786E+00 B=  0.14750E+00  COVARIANCE  5./  TEST FOR F,  1/  TEST FOR F,  1/  OVERALL F.  2./  5.=  0.127E+02  SLOPES 10.=  O.140E+03  LEVELS 11.=  0.772E+01  TEST 10.=  CORRELATION COEFFICIENT  SEY=0.8545E-01 SEY=0.2401E-01  R= R=  0.9895 0.9786  DF= DF=  6. 6.  SEY=0.2896E+O0  R=  0.7400  DF=  13.  ANALYSIS  TEST FOR HOMOGENEITY OF RESIDUAL F,  STANDARD ERROR OF ESTIMATE  O.123E+03  VARIANCES  DEGREES OF FREEDOM  L I Q U I D COMPOST  LINE  1 : B+C DAY  LINE  2  : CHAE DAY  INDIVIDUAL SIMREG  1 2  A= A=  0.35857E+00 0.57286E+00  SIMREG FOR POOLED A= 0.46571E+00  STANDARD ERROR OF ESTIMATE  CORRELATION COEFFICIENT  B= 0.15929E+00 B= 0.48214E-01  SEY=0.4890E-01 SEY=0.2401E-01  R= R=  0.9917 0.9786  DF= DF=  6. 6.  B=  SEY=0.1762E+CO  R=  0.7861  DF=  13.  LINES  DATA 0.1O375E+O0  COVARIANCE  ANALYSIS  T E S T FOR HOMOGENEITY OF RESIDUAL VARIANCES F.  5./  T E S T FOR F,  1/  T E S T FOR F,  1/  OVERALL F,  2./  DEGREES OF FREEDOM  5.=  0.415E+01  SLOPES 10.=  0.116E+03  LEVELS 11.=  0.109E+02  TEST 10.=  O.121E+03  APPENDIX B  Statistical analysis of mushroom yields.  VARIABLE  NAMES  DATA  - YIELD  FORMAT  (I2.4X.F6.1)  CONVENTIONAL  YIELD  ANALYSIS OF VARIANCE SOURCE TREAT ERROR TOTAL  GRAND MEAN  DF  SUM  3 7 10  14500. 2763.4 17264.  MEAN  SQ  ERROR  PROB  F-VALUE 12.244  4833.4 394.77  0.35742E-02  144.60  STANDARD D E V I A T I O N OF  FREQUENCIES.  SQ  - YIELD  VARIABLE  MEANS. STANDARD  1 IS  41.550  DEVIATIONS  ******************************************************************************** TREAT MN  YIELD  1  2 172.2  3 170.8  4 138.0  73.80  STUDENTIZED RANGES FOR NEWMAN-KEUL'S T E S T , ALPHA=0.05 3.344 4.165 4.681 THERE ARE 2 HOMOGENEOUS SUBSETS FOR A SUBSET OF THAT S I Z E ) WHICH ARE ( 4 ) ( 3, 2, 1) TIME  FOR  ANALYSIS  MULTIPLE  COMPLETE.  RANGE T E S T S IS  (SUBSETS OF ELEMENTS. L I S T E D AS FOLLOWS  NO  PAIR OF WHICH D I F F E R BY MORE THAN THE  SHORTEST S I G N I F I C A N T RANGE  0.1180E-01' SECONDS.  OJ  VARIABLE DATA  YIELD  NAMES  - YIELD  FORMAT  - TWO  (I2.4X.F6.1)  PERCENT  MALT  ANALYSIS OF VARIANCE SOURCE TREAT ERROR TOTAL  GRAND MEAN  DF  SUM  SO  MEAN SO  3 8 11  27. 189 15.893 43.082  9.0631 1 .9867  ERROR  PROB  F-VALUE 4.5619  0.38232E-01  2.1250  STANDARD D E V I A T I O N OF  FREQUENCIES,  - YIELD  VARIABLE  MEANS, STANDARD  1 IS  1.9790  DEVIATIONS  ***************************************************************** 3  TREAT MN  YIELD  1 .500  4 3.933  3.067  0.0  STUDENTIZED RANGES FOR NEWMAN-KEUL'S T E S T . ALPHA=0.05 3.261 4.041 4.529  THERE ARE 2 HOMOGENEOUS SUBSETS FOR A SUBSET OF THAT S I Z E ) WHICH ARE ( 4. 1, 2) ( 1. 2, 3) TIME  FOR  ANALYSIS  M U L T I P L E RANGE T E S T S IS  COMPLETE.  (SUBSETS OF ELEMENTS, L I S T E D AS FOLLOWS  0.8255E-02  NO  PAIR OF WHICH D I F F E R BY MORE THAN THE  SHORTEST S I G N I F I C A N T RANGE  SECONDS.  -•4  VARIABLE DATA  YIELD  NAMES - Y I E L D FORMAT  (I2.4X.F6.1)  - L I Q U I D COMPOST  ANALYSIS OF VARIANCE SOURCE  DF  SUM  4 10 14  444.98 47.587 492.57  TREAT ERROR TOTAL  GRAND MEAN  MEAN  ERROR  SQ  PROB  F-VALUE 23.377  111.25 4.7587  0.46429E-04  9.6733  STANDARD D E V I A T I O N OF  FREQUENCIES.  SQ  - YIELD  VARIABLE  MEANS, STANDARD  1 IS  5.9316  DEVIATIONS  ******************************************************************************** TREAT MN  1  YIELD  2  9.000  3 12.30  4 10.57  5 16.50  0.0  STUDENTIZED RANGES FOR NEWMAN-KEUL'S T E S T , ALPHA=0.05 3. 151 3.877 4.327 4.654 THERE  ARE  3  FOR A SUBSET ( 5) ( 1. ( 4) TIME  FOR  HOMOGENEOUS SUBSETS OF  (SUBSETS OF  THAT S I Z E ) WHICH ARE  3.  ELEMENTS,  NO  PAIR OF WHICH D I F F E R BY MORE THAN THE  SHORTEST S I G N I F I C A N T RANGE  L I S T E D AS FOLLOWS  2)  MULTIPLE  RANGE TESTS IS  0.8568E-02  SECONDS. -~4 tn  ANALYSIS  COMPLETE.  

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