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tWDE OF ACTION DOTHISTROl'UN
A Thesis pros en ted in pn.rtic.l fulfilncm t of the requirenents for tho dogreo of Doc tor of PLilosoph:-,-, Jlilc.ssoy University,
New Zer�lnnd
A .M.. Harvoy
July, 1974.
ABSTHLCT
Dothistrocin is c. bright orrmge-rcd pignont produced by the pino-neodlo p'1thogen Dothistronn pini Hulbnry, the cnuso.l ae;ent of 1': necrotic cl:.sonse lmovm as dothistrouo.l blight. This
conpound, ioplico.t&d o.s o. fungal toxin, has been isolo.ted both fron lQborntory cultures of D.pini e..nd fron inf&ctctl Pinus r::-.dintu.
foliage.
Detailed cheLtical investigation by Ga.ll�tghDr
(
1 971 ) shovredthnt dothistrooin is o. tri -ct- hydroxyr:mthrnquinone fusocl to o.
s�bstitutod tetro.hydro�ifure..n rine; systen. Tho bifurnn ring ooioty is incorporated in othvr fungal netnbolitos including the oxtrenoly toxic and co.rcinogcnic a.fla.toxin cocpouncls. Thoro is o.n increasing body of evidcnc0 to suggest tho.t these fung�l oot::-.bolites sho.ro c. co�on biosynthetic origin.
Bion.ssD.y has do;-Jonstrntotl tho toxicity of dothistronin to the unicellular green clgn, Chlorollo. pyrenoidoso., nnd to tissue cultures of P. a tte"'::tm to.. The vory lmv level of solubility of the conpound in aqueous solutions ha.s precluded bionsso.y using pine seedlings. This thesis reports o.n investigation of the biochenico.l chnngos induced by do thistronin in ni crobiological systens.
iii.
In the course of this inv�stigntion dothistronin hns boon shown to bo toxic to n rnnge of uicroorgo.nisns in addition to its known toxic i ty to Chlorollo. pyronoidosn. Tlwsu studies hc.vu sugr:osted
possible wnys of incro:J.sing tho sensitivi ty of bio::ctwc.ys for
dothistronin.
It 1n:.s found th<'.t tho ncldi tion of dothis tronin to J.iquid cultures of Chlorelln ::cs r:m uthyl �'.cotnto solution cnusod.
re1)roduciblo levGls of inhibition, prov ided thnt the ethyl e .. cotn.te concontrntion 1-rns loss them 0.5)b. Bo.tch culture techniques ivore used to estnblish the lovels of dothistronin required for
inhibi tion of growth of Chlorelln. Thu rntio of dothistronin concentro.tion t o coll nm1be r wns found to bo nn iuportnnt fac tor in tho inhibi tion rosponso.
Utilization of synchronous culture techniques pc.:rr.littod the s tudy of biochcr.ricnl cho.ngos induced by dothistronin throu.shout
tho coll cycle of Chlorolln. Results showed c. nnrked inhibition of the rnto of increo.se of totnl protein nnd RNA over the cell cycle \vi th no significant .:'.1 h::rntion of the rnto of DNL incroc.se.
A dose-response curve for dothistrooin inhibition of grm.rth of Chlorelln vms ostnblished "-nd Q norc detailed invostie;o..tion of tho ac tion of dothistronin in inhibiting grovith uns undertaken using rndioo.c tive i sotopes. By this nenns i t was shown that 3H-uridine r�d 14c-phenylnlnnino incorporation into cell untGrinl
is inhi bi tod vri thin 30 nins of exposure to the toxin. Difficulties encountered in a ttor.1pts to ol)tr.,in so. tisfnctory incorpor.:c tion cf le. bel into Chlorella DIITA-fro..ctions :tJrcvonted further inv0s tic;·'"' tion of the effect of dothistronin on DNA synthesis in this organiso. This led to tho investigation of other nicroorgnnis�s as noro suitable
experinontnl systens f 'r this study.
Bacillus negntoriuo
��,
proved to bo very sensitive to dothistronin n.nd showed rnpid incorporo.tion of radioactiveisotopc8 into protein o.nd nucleic �cid fractions. Grm,rth curves ostnblished thnt the inhibitory r."ltios of dothistronin concentration to cell nunbers for this orgnnisE woro in the order of 0.25)4S/cell
X
108(o.s cocpo..red to 2.0�tg/ccll X 108 for Chlorclln). At this concen- tro.tion, over the 30 nin tine course studied, dothistronin ho.d no effect on the incorporation of 3H-thynidino into the DNA frc.ction.
Inhibition of 3n-uridine incorporntion w:J.s evident ,'lt 6 oin and very onrked by 10 oin while the inhibition of 14c-phenylnlanine incorporation into protein 1ms not evident until considcm::cbly lnter.
The effects of dothistronin in this systeu ·wore
conp:cre!11'!i
th thosoof o..ntibiotics vri th kn0
1
m si t€6 of action. Dothistronin inhibition of 3H-uridino incorporation hns n sinilnr tine course to tho.t shown by actinonycin D, nlthough narked inhibition by .0ctinonycin D is evident nt 3 Din, whereas dothistronin inhibition is not noticeable until6
nin.v.
On the basis of these results it i s suggested that
do thistromin interferes >vi th RNA syn thesis and that the observed.
inhibition of protein synthesis i s a s econdary effect of this impairment.
Confirmation of dothi3tromin action 'in situ'by administration of the c ompotmd to pine seedlings i s necessary before any definitive s tatement can be made concerning its role in dothistromal blight.
However these results indicate the po ssible importance of
dothistromin in pathology of dothi stromal needle blight of pines.
Impairment of the RNA synthetic capacity in pine need l e tissue by the toxin could rapidly lead to cell death and to the necrosis of needle ti ssue observed in diseased foliage.
ACK.!�OULJ.i.!DG EiviEIJTS
I >vish to express my thanks to the follm<ling people:
tfy supervisors, Professor R.D. Bntt o.nd Dr. G.G. Pritchnrc'., for their holpful discussion o.nd oncourn.gonent during the course of this work.
The '1o.ny othur ncnbors of the ncndonic nnd technical stc.ff 0f tho Depo.rtucnt of Chonistry, Biochenistry nncl Biophys ics o.nd the Depnrtnent of MicrobiolOGY, i<lho hnvo c-ivon freely of their tico for useful discus sion, nssis tnnc._, o.ncl
encourngenent.
Dr. C. Bnssett <.:nd s tnff of tho Forest Research Institute, Rotorun, for help o.nd advice on aspects of this thesis.
TABLE OF CON'l'ENTS
ABSTRACT
A CKN01tTLEDG EMENTS TLBLE OF CONTENTS LIST OF FIGURES LIST OF TABLES
1 • 1 .1
1 . 2
INTRODUCTION
DOTHISTROMAL PINE BLIGHT
1. 1 1 The history of dothist ronnl blight
1 .12 The nature of the disease
1 . 1 3 The control of the dise�se
DOTHISTROMIN, .A SECONDLRY NETABOLITE OF D.PINI
vii.
ii.
vi.
vii.
xiii.
xv.
1.
1 •
1 •
2.
4.
5.
1 .22 Possible rel.:tionships with other nycotoxins
6.
1 . 221 The afldoxins 8.
1.222 Oth0r fung�l netabolitos containing bifuran
noieties. 1 1 .
1.23 Possible structure - nctivity relationships for dothistro�in.
1 .3 AIMS OF THIS INVESTIGATION
2. EXPERIMENTAL METHODS
2. 1 ORGANISMS
2. 2 MATERIALS
1 4 .
14.
1 5 .
1 5 .
1 5.
2 . 3
2.4
I SOLATION .A.ND PURIFICATION OF DOTHISTROMIN 2 . 3 1
2.32
2 . 33 2.34
Laborc.tory culture of D.pini Extrnction of dothistrooin Purification of dothistrooin
Precaution· .ry nc;csures for handling dothis tronin
MICROBIOLOGI CAL HETHODS
2.41 Mainten�ncc of Chlorolla culturos
2 . 42 Liquid culture of Chlorolla
2.43 Use of Chlorella in plate bionssey
2 . 44 Bioassay procedure using other nicro-orgnnisos
2.45 Nec.sureoent of growth of Chlorella in
liquid culture.
2.46 Synchronous culture of Chlorella.
2 . 461 Introduction to synchronous culture techniques.
2 . 462 The cell cycle of Chlorella
2.463 The developoent of tecru1iquos for synchronous culture of Chlorella.
2 . 4631 Prolioin��.ry studies.
2.4632 Procedure for obtaining synchronous cultures of Chlorolla.
1 8.
1 8.
1 8.
20.
2 1 . 2 1 •
2 1 •
22.
24.
2 5.
2 5 .
2 5 . 29.
32 . 32.
35 . 2.464 Assessoent of degree of synchrony achieved. 37 . 2 . 47 Maintenance and
2.48 Maintenance and
2 . 49 Measurenent of B.negateriun.
growth of S.cerevisine.
growth of B.oegnteriuo.
growth of S.cerevisine and
41 . 41 .
41 .
2.6
2 . 7
ANALYTICAL liETHODS
2 . 51 Detornin': tion of nucleic ncids nnd protein
2 . 5 1 1 Modified Schnid t-Thnnnh[�user extraction
procedure.
2.512 Ribonucleu.so digestion of RNA
2.5 1 3 Esticntion of nucleic ncids
2.514 Estin�tion of totnl protein
2.52 Fluorinetric detection of dothistronin.
Rl\DIOCHEMICAL NJ�THODS
ix.
42. 42.
42.
4 4.
45. 45. t�6.
48. 2.61 Scintill�tion counting procedure 48.
2.62 Count corrections 48.
2.63 Lnbelling procedure for Chlorella. 49.
2.64 Labelling procedure for S.corevisino nnd
B.negateriuo. 50 .
2 . 65 Investigntion of the distribution of isotopic
label nnong cell fractions. 5 1 .
2.65 1 Electrophoresis of RN1, hydrolyzt>.te. 52.
2.652 Chronntography of DNA hydrolyzate. 53. 2.653 High voltngo electrophoresis of protein
hydrolyznte. 56.
MICROBIOLOGICAL UEDIA 58 .
3.2
EXPERIMENTAL RESULTS
GROWTII INHIBITION OF MICRO-ORGANISHS BY DOTHISTRmUN
3 . 11
3 . 12
Scrooning of nicro-orgnnisn3 for dothistronin growth inhibition.
Al tt;rn:c ti ve bioasscy DG thod.
3 . 1 3 Detection of dothistrouin in zonos of inhibition.
3.14 Re l�tive toxicity of other hy dro):yr:.n thr.�.quinonos.
3. 1 5 Assessuent of tho bionssny.
IN1UBITION OF GRQ1<lTH OF' Ci:ILORELLA IN LI�tUID CUL'l'URE.
3 . 21 Problens cssocinted with the low solubility of dothistroDin.
3.22 Effect of dothistro;Jin on grovrth of Chlorelln in bn tch cul tur(�S.
3.221 Effect o f nddition e t tho beginning of the
63.
63.
65.
68.
71 .
76 . 78.
80.
81.
82.
growth cycle. 82.
3.222 Effect of nddition during the log-phnso of
tho growth cycle. 83.
3.23 Effoct of dothistronin on Chlorella in
synchronous culture. 86.
3.231 The cell cycle of Chlorella. 86.
3.2 32 Effect of dothistrosin on cell division,
nucleic acid end protein le ve ls in Chlorelln 88.
3. 24 Sur.mary nnd discussion 96.
EFFECT OF DOTHISTROMIN ON THE INCORPORATION OF R.ADIOJ;,CTIVE PRECURSORS BY CHLORELLA
3 . 31 Incorporation of rndionctive precursors by
Chlorella cultures.
99.
102.
3. 32 Effec t of dothistro�in on precursor incorpor:� tion.
3 . 33 Effect of do this troDin on pr::;cursor incorporn tion c. t difforen t s t<:ges of the cell cycle.
xi.
103.
104. 3 . 34 Rel�tionship between dothi stronin
c oncentr;1.tion end incorporn. ti on of 3H-uridino. 109.
3 . 35 Dis tribution o f precursor lnbel in cellulo.r frnc tions.
3. 36 Ll torn::tive lc.belled precursors for mu.
synthosis.
EFFECT OF DOTHISTRONIN ON GH.OHTH LND PRECURSOR INCORPORJ.TION BY YE; ... ST J.ND B.MEGL'rERiill'!.
11 o.
114 .
115.
3.41 Inves tigntion of S.cercvisine (Yens t ) 1 16.
3.411 Effec t of dothi stror.'.in on growth of yeas t. 116.
3.1).12 Incorpor:� tion of lnbelled precursors by yer:st.
3.42 Invcs tigntion of B.uegr'..teriun IG1.
3 . 421 Effect of dothistronin on growth of B.negnteriun KH.
118.
121 •
122 . 3. 422 Uptnko of precursors by B.negnteriun KM. 122. 3. 423 Prelininnry investigr>. tion of precursor
incorporr>. tion in thv presence of
dothis tronin. 126.
3.43 Conpnrison of effec ts of dothis tronin nnd antibiotics on precursor incorporntion in
B.ncgntcriun KM. 133.
3.44 Conparison of effects o f dothis tronin with
thoso of rclnted conpounds. 143.
4.
4.2
DISCUSSION �ND CONCLUSIONS
GR01·1TH INHIBI TION OF HI CRO-OnGfJITISMS 4. 11 Lg�r-plnte bioGss�y.
4. 1 2 Inhibition of nicro-o:rgo.nis!JS in liquid cultures.
4.13 Ioplicc..tions for potGnti:ll quGnti tGtivo bionss;::.y.
CELLULi.R SITE OF DOTHISTROrUN INHIBITION
1 48.
148. 149.
1 5 1 • 1 54.
1 56.
4.2 1 Sito of inhibition in Chlorelln pyrenoidosn. 1 57.
4.22 Site of inhibition in B.ncgo.toriun. 1 59.
4.221 Validity of precursor o.ppronch. 160.
4. 222 Dothistronin inhibition of precursor incorpor£: ti on.
4.23 1-iode of .'lotion of structuro.lly relnted nycotoxins.
4.24 il.ro:..'..s for further investig:.l.tion.
163.
1 64.
1 70.
4. 3 ROLE OF DOTHISTRONlN IN DOTHISTRONJ.L PINE BLIGHT 171 .
4.4 DOTHISTROMIJIT :.s L. POTENTH.L HEALTH Hi.Zi.RD. 1 76.
REFERENCES
Figure
2 3
4
5 6 7 8 9
10 1 1 12 13
1 4 1 5
16
17 18
xiii.
LIST OF FIGURES
Coopounds isol:'..ted frou D.pini cultureii. ?.
Lflntoxins �nd co-2et�bolites 9.
The vcrsi colorin nnd s tGrign�tocystin
groups of fungnl net::1bolites. 13.
Scheo�tic di ngrno of culture nppnrntus
for Chlorclln. 2 3.
Stnnd.::1.rd curve for Chlorell.s.. 26.
The 1Tnniya1 cycle. 30.
Cell size di stributions. 34.
Fluores cence exci tation and eni ss ion spectra. 47.
Electrophoresis of IDJL. hydrolysate. 54 .•
Chronfl. togrephy of DNL hydrolys<"'.te. 55 . Electrophoresi s of p rotein hydrolysate. 57.
St�ndr>.rd CUrve for I dothistrODin ,"..gClrS I o 73o Growth curves for Chlorelln showing
inhibitory affect of dothistrooin. 84.
Gro\vth curves for Chlorello. showing
inhibitory effect of dothistrooin. 85.
Cell counts o.nd cell s ize di stributions over one cycle of synchronous cultures of
Chlorella. 89.
Changes in level s of oacroooleculnr
constituents ove r one cycle of synchronous
cultures of Chlorelln. 90.
Effect of dothi stronin on the cell cycle of
Chlorello.. 92.
Effe ct of dothistronin on synchronous
cultures of Chlorella. 93.
Figure
19 20 21 22
23 24 25 26
27 28 29
30 31 32
33 34
35
36
Effect of dothistronin on synchror.ous cultures of Chloroll.".
Effect of dothistror:in on synchronous cultures of Chlorelln.
Effect of dothistro;.lin on the incorporation
Pnge
94.
95.
of lnbellGd precursors by Chlorelln. 105.
Rntes of htbel incorporntion over tho coll
cycle of Chlorella. 108.
Dose-response curve for dothistronin. 111.
Growth curves for Ye.�st shm-T inhibitory
effect of dothistromin. 117.
Growth curvo for B.negatc::riun KM sho\'l'ing
inhibitory effect of dothistronin. 123.
Effect of proincub�tion on dothistronin
inhibition of 3H-uridine incorporation. 127.
Cooparison of assay techniques. 130.
Prelininnry labelling of B. oegn toriun KN. 131.
Growth curves of B.negnteriun ��
estl=tblishing growth inhibitory
concentrations of �ntibiotics. 135.
Effects of dothistronin nnd chlornnphenicol on pro cursor incorporD .. tion.
Effect of dothistronin and actinoi:lycin D on precursor incorporation.
Effect of dothistroLrin and nitonycin C on precursor incorporation.
Structures of fungnl notnbolites.
G�wth curves of B.no,9;c.teriun KIJI
es tablishing growth inhibitory
concentrntions of fungal netnboli tos.
Effect of nflntoxin and s terignntocystin precursor incorpor<ltion.
Sites for inhibi tion of nucleic acid synthesis by antibiotics and drugs.
on
137.
139.
141 • 144.
145.
147 0 172.
Tnblo
2 3
5 6
7 8 9
LIST OF TABLES
Methods for obtnining synchronous cultures.
Synchronization indicos.
Growth inhibitory activity of dothistronin on
n selection of oicro-orgnnisos.
Bioassny by direct application of toxin prepnration.
FluorinQtric nnnlysis of bionssay plates.
Disc bioassny of anthraquinone toxicity to Chlorelln.
Dothistrocin cell nunbor r:l tios.
Suns,�ry table: Effocts of dothistrouin on synchronous cultures of Chlorolla.
Rnte of lc.bol uptnke ovor life cycle of Chlorelln.
10 Distribution of label in collulrrr fractions of Chlorelln.
11 Label distribution in cellular fractions of Yc�:tst.
12 Distribution of lnbol in collulnr frnctions of B.nego.teriun.
13 Inhibition of 3H-uridine incorpor<'. tion by increasing concentrations of dothistronin.
xv.
27/28.
40.
70.
75.
77.
87.
97.
107.
11 3.
120.
125.
132.
