Studies on the Structure and Functions of Lycopersin, a Pigment From Fusarium lycopersici and Fusarium vasinfectum

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FORDHAM UNIVERSITY GRADUATE SCHOOL

Magr 15

19.50....

This dissertation prepared under my direction by

.............. C&r&l&Kreitmaii.........................

entitled „STOMS.QN..^...^^ A PIGMENT FROM FUSARIUM LYCOEERSICI AND FUSARIDM VASINFECTDM

has been accepted in partial fulfilment of the requirements for the Degree of...... Doctor of Philosophy.. .................. .

Dr. F r i e d r i c h ..... (Faculty A d vise r)

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STUDIES ON THE STRUCTURE AND FUNCTIONS OF LYCOPERSIN, A PIGMENT FROM FUSARIUM LYCOPERSICI AND FUSARIUM VASINFBCTUM

BY GERALD KREITMAN A.B., BROOKLYN COLLEGE M.S., FOREHAM UNIVERSITY

tJjB

DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF CHEMISTRY AT FORDHAM UNIVERSITY

NEW YORK

1950

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ProQuest N um ber: 10992947

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uest ProQuest 10992947 Published by ProQuest LLC(2018). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

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TABLE OF CONTENTS

LIST OF TABLES

......

Page ill

LIST OF FIGURES '.............................. ACKNOWLEDGEMENTS

iv

.........................

▼

I.

INTRODUCTION..........................

1

II.

METHODS

6

a) b) c) d) III.

.........................

Microbiological •••••••.••••••.... Analytical...... ...... Spectroscopic determinations •••••••• X-ray diffraction ••......

EXPERIMENTAL.......................... a) Conditions for growth of Fusarium lycopersici .... ............ . b) Conditions for growth of Fusarium vasinfecturn ♦................ ..... c) Isolation of lycopersin ••.••.•••••.. d) Structural study of lycopersin e) Effect of lycopersin on fat formation in Fusarium lycopersici f) Effect of lycopersin on fat formation in Fusarium vasinfecturn...... g) Antibiotic activity ........ h) Mechanism of formation of lycopersin ......

6 8 12 15 16 16

18 22 28 hi $6 ol 61}.

IV.

DISCUSSION ............................

69

V.

SUMMARY.....................

76

BIBLIOGRAPHY............

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LIST OP TABLES

Table I. II* III* IV* V. VI.

VII*

VIII. IX.

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Page pH changes on Raulin-Thom and Czapek-Dox media ••»•••••.......

20

The effect of temperature on the growth of Fusarium vasinfectum ......

21

Extraction solvents for lycopersin*•••••••

2tg.

Color changes of lycopersin with alkalies ••••••••••*..•••.*...... ••••

31

Analytical data of lycopersin ••«••*......

32

Effects of riboflavin and nicotinic acid on fat formation in pigmented Fusarium lycopersici ••••••..... •••••*••••

1+9

Effects of riboflavin and nicotinic acid on fat formation in unpigmented Fusarium lycopersici •••*•«........ *••••••

fij-

Changes in the composition of fat in Fusarium vasinfectum •••••••••

59

Trapping experiments .............

68

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LIST OP FIGUR3S

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Figure 1.

Interference of ergosterol in spectral determination of linolenic acid •••••••••••

10

2.

Standard curve of lycopersin ............

13

3.

Ultraviolet absorption spectrum of lycopersin .••••••••.... *............

27

ij.. 5.

Ultraviolet absorption spectrum of . lycopersin diacetate •••••••.

•

35

Ultraviolet absorption spectrum of lycopersin dibenzoate ••••••••..........

37

6.

X-ray powder patterns of lycopersin.....

39

7.

Infrared absorption spectra of lycopersin and lycopersin diacetate •••••••

I4-I

Infrared absorption spectra of lycopersin and lycopersin diacetate .......

hfi

Infrared absorption spectra of lycopersin and lycopersin diacetate

k-3

Ultraviolet absorption spectra of oils of pigmented Fusarium lycopersici .........

51

Ultraviolet absorption spectra of oils of unpigmented Fusarium lycopersici •••••

53

Ultraviolet absorption spectra of isomerized oils of Fusarium vasinfectum .................

62

Ultraviolet absorption spectra of free oils of Fusarium vasinfectum

66

8*

9. 10* 11. 12.

13.

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Page

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ACKNOWLEDGEMENTS

The author wishes to express his gratitude to his wife and parents for their continued inspiration, encouragement, and devotion.

This investigation was carried out under the auspices of the Office of Naval Research and aided by a grant of the P. G. Cottrell Fund of the Research Corporation.

This study was carried out tinder the direction of Dr. P. P. Nord.

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STUDIES OK THE STRUCTURE AND FUNCTIONS OF LYCOPERSIN, A FIGMENT PROM FUSARIUM LYCOPERSICI AND FUSARIUM VASINFECTUM

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1. INTRODUCTION PREVIOUS INVESTIGATIONS WITH FUSARIA Among the Pungi 3inperfecti (Deuteromycetes), the members of the genus Fusarium have attracted the attention of investigators for many years. These molds are of con­ siderable industrial importance. As plant pathogens they are found on every continent, and they are known to attack practically every type of commercially important crop, e.g., cotton, flax, tobacco, rice, tomato and banana. The tomato wilt disease, also known as Fusarium wilt and sleepy disease (112), was described in 1905 (92), and Fusarium lycopersici (link.) was identified as the causa­ tive agent. In 1922 lj.00,000 tons of tomatoes were destroyed by this fungus (102), and its devastations have continued unabated until the present t ime. The problem has been attack­ ed from two directions. Since the development of wilt-resist­ ant tomatoes (102), tomatin (lycopersicin) has been isolated from the tomato plant and found to be active against the spores of Fusarium oxysporum (57)* Investigations on the nature of the wilting agent showed that the effect was produced by a toxin (66). Plattner (99) succeeded in iso­ lating the active principle, which he named lycomarasmin, And Woolley (139) proved it to be a tripeptide of aspara­ gine, glycine, and a-hydroxy alanine. It was also shown that the wilting effect was due to permeability changes in the plasma membrane and the water-holding capacity of the

2

‘plasma itself (l{-3). A second series of investigations have explored the Fusaria in the hope of finding among them suitable sources for new antibiotics* The bacterial spectra of both the mycelia and culture filtrates of Fusarium bulbigenum and Fusarium oxysporum have been determined and found to be active against Gram positive bacteria (123), The antibio­ tics isolated from several species of Fusaria have exhibit­ ed .in vitro inhibition of the growth of Mycobacterium tubeycnlnaia (33533a). Recently it was shown that enniatin A, obtained from Fusarium orthoceras, was identical with lateritium I (100). Other Fusarium antibiotics were found to be different from each other, but to have a similar chemical group (2 3 ). The Fusaria have been the subject of a third series of investigations of a more fundamental nature. Based on the concept that only unaltered living systems can serve as a means to elaborate the "spectrum” of the cell, their alcoholic fermentation of hexoses and pentoses was studied (82,81).,85,11151335134)• The presence in Fusaria of a power­ ful dehydrogenase has been demonstrated (1)5 ,106 ) and these molds have been shown to possess enzyme systems capable of dehydrogenating fatty acids (79). Among the vitamins, thia­ min (111) and riboflavin (90) are present in the mycelium. Characterized as powerful fat formers (79,81,83), their fat has been shown to be similar to olive oil (80). From the unsaponifiable fraction, ergosterol was isolated (3 8 ).

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This work with the Fusaria fa^s has been extended by the

isolation of an active lipase preparation (3 8 )# Present enzymatic investigations with Fusaria have been directed at the elucidation of the possible functions and of the mechanism of formation of the pigments so charac­ teristic of these molds. Despite the observation of the photodynamic effects of the fluorescent dye, hypericin, on protozoa in 1909 (121), very little work had been concerned with the Fusaria pigments* Considered as "waste products11, the only interest in these coloring matters was as a means of classification of the molds (16 ,115 ,136 ,13 7 ). Viflaile some of the pigments had been extracted from Fusaria (9>l6) only a few were actually isolated. Bexssonoff (10) reported the isolation of two pigments from Fusarium orobanchus* while Raistrick and coworkers studied the structures of rubrofusarin and aurofusarin, which they had isolated from Fusarium culmorum (5)* Two closely related substituted naphthoquin­ ones have been isolated from Fusarium javanicum by Arnstein and his associates (3*li-)* An intensive investigation of the pigments of Fusaria was undertaken by Mull and Nord (80,86), who succeeded in isolating both rubrofusarin and aurofus­ arin from Fusarium graminearum Schwabe* By a comparison of the absorption spectra of related xanthones with that of fubrofusarin, they proposed that the latter was either 2,8dihydroxy, 1-methoxy,7-methyl or 2,3-dihydroxy,8-methoxy,7methyl xanthone. Weiss and Nord (127*129) obtained a new gigment, solanione, by petroleum ether extraction of the

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mycelia of Fusarium solanl D2 purple, This mold was report­ ed to form other pigments (130 ), some of which were excreted into the media, while others were retained in the mycelium* On the basis of certain chemical reactions and degradation studies, as well as infrared and ultraviolet absorption spectra, the structure of solanione was thought to be 2 methyl,3 -methoxy,6 or 7 -&cetonyl,5 »8 -dihydroxy,l,lp-naphthoquinone* That natural pigments may intervene in the enzymatic processes of the living cell has been amply demonstrated*By means of these compoilnds a multitude of effects have been produced in living organisms, for example: an increase in the rate of respiration by bacteria (ip2); a decrease in the rate of dehydrogenation by molds (86,110); and an inhibition of certain enzymes (53*56,12ip). It has been shown that a red pigment from cereal seeds can act as an oxidation catalyzer in carbohydrate metabolism (lp9), and it has been claimed that the carotenoid pigments of certain yeasts may possibly participate in the respiration processes of the cell (6 7 )* Previous investigations have shown that the addition of sol­ anione to a growing culture of Fusarium lini Bolley* which does not form a pigmented mycelium, results in a decrease In mycelial weights (128), a lowering of the carbohydrate con­ version factor (8 9 ), and an increase in the desaturation of the fats (28). Similar effects were produced by nicotinic acid and riboflavin (90)* In addition, it was observed that jthe amounts of sterol formed by this mold when grown in the -*

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presence of naphthoquinones related to solanione were effected (69)* Enzymatic investigations with pigmented Fusarium lyco­ persici showed that its behavior was different from that of the non-pigmented members of this genus* Although the fungus increased its fat and pigment formation with increased glu­ cose concentration in the medium (8 9 ), yet solanione had no effect on its fat formation (2 8 ). These reports aroused our interest in the possible role of the pigment in the carbohydrate— >fat conversion. There­ fore an investigation of the structure, function, and mode of formation of the pigment was undertaken. Since Fusarium vasinfectum exhibited similar growth patterns, it too was included in this study.

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II. METHODS a) MICROBIOLOGICAL METHODS Cultures Etaployed The following Fusaria were used as enzyme sources in these experiments: Fusarium lycopersici R-5-6, obtained from the XT. S. Department of Agriculture, B©itsville, Maryland, through the courtesy of Dr* S. P. Doolittle; Fusarium lyco­ persici-2, obtained from the University of Tennessee through the courtesy of Dr. C. D. Sherbakoff; Fusarium vasinfectum F-273, obtained from the New York Botanical Garden through the courtesy of Dr. Wm. J. Robbins. Fresh isolates of Fusarium lycopersici were prepared from tomato plants of the Rutgers variety which were infect­ ed by dipping their roots in a suspension of spores of the fungus (31). The ability to produce wilting of tomato plants was used as the chief basis for classification. The stock cultures of Fusarium lycopersici were main­ tained on potato dextrose agar medium, while those of Fusar­ ium vasinfectum were kept on malt agar. All cultures were transferred at two week intervals. Because of numerous reports of changes in pure cultures of Fusaria (5l>75*119) and of the different growth character­ istics of single spore inoculations (5 0 ), test inoculations were made prior to large-scale cultivation of the molds to determine whether they maintained their typical growing appearances on liquid media. In the case of Fusarium lyco­ persici it was found that as few as three consecutive

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transplants of the fresh isolates resulted in the loss of

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the ability to produce pigment. The fully grown mycelium was colorless except for isolated patches of pigment. This form of themold has been considered as the "unpigmented11 form, while

the typically heavily pigmented mycelium result­

ing from inoculations prepared from fresh isolates has been referred to asthe "pigmented” form. Cultural Conditions A.

All preliminary experiments to determine the opti­

mal conditions for growth and pigment production (pH, temper­ ature, carbon source, trace elements and light) were con­ ducted in 125ml. Erlermieyer flasks, each flask containing 25 ml. of nutrient medium. Either Raulin-Thom or modified Czapek-Dox media were used : Raulin-Thom medium (89) ij..00g. l^.OOg. 0 .60 g. 0 .60 g. O.i^Og. 0 .25 g* 0.07g. 0 .07 g. 1500 ml.

tartaric acid ammonium tartrate potassium carbonate dibasic ammonium phosphate magnesium carbonate ammonium sulfate zinc sulfate (7H?0) ferrous sulfate T7H2O) tap v/ater

Modified Czapek-Dox medium (7 8 ) 8.35g. 8.35g. 0.8l>.g. 0.03g. 0.003g. 1000ml. B.

sodium nitrate potassium phosphate (primary) magnesium sulfate (7H0O) ferrous sulfate (7H2O) zinc chloride distilled water

Large-scale experiments for the purposes of ob­

taining pigment or oils were carried out in 3-1• Fernbach

8

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flasks, each containing one liter of Raulin-Thom nutrient

*

medium. Fusarium lycopersici was also cultivated on a larger scale in special Pyrex trays (30xlj.5x8 cm.) placed in a sterilincubator (2 7 ) holding ll| trays, each tray containing 3 liters of nutrient medium* C.

Sterilization of the medium in the Pyrex tray£

was accomplished by streaming steam through the sterilincu­ bator for a thirty-minute period on three successive days* The media contained in flasks were sterilized for twenty minutes at 120°C. and under 15 pounds pressure. When alkali was to be added, it was sterilized separately and then added with aseptic technique to the sterile media. This prevents excessive decomposition of carbohydrates. D*

Inoculations were made by means of uniform spore-

mycelial suspensions prepared from the agar stock cultures by the methods previously used in this laboratory (ip6,133>135)* The Fusaria were incubated at the optimal temperature determined in the preliminary experiments, or were grown in a dark room whose temperature was kept close to the optimum by meansof an electric heater and fan. b) ANALYTICAL METHODS Fats The constants for the fats were determined according to the methods of the Association of Official Agricultural Chemists (71)* The method for the spectral analysis of the isomerized fatty acids is an improvement of that suggested by Brice andj

9 p

Swain (13)* These spectral analyses are based on the absorp­ tion peak of linolenic acid at 268 -2701pC( (15,76*77)* However in the vegetable fats, e.g., those of Fusaria* the presence

of ergosterol, which has one of its absorption maxima at 2?1 HJitKSL1-), would definitely interfere in these determinations* The preparation of reagents and the isomerization pro­ cedure are identical to those described (13)* The treated sample was then dissolved in 50 ml. of methanol and 100 ml. of water added. This solution was extracted with three 100 ml. portions of ethyl ether to remove sterol, acidified with % S 0^, and again extracted with three 50 ml. portions of ethyl ether. After the ether extract was washed free from acid with water, it was dried with anhydrous Na2 S0j^ and filtered through a sintered glass funnel. The residue obtain­ ed after removal of the solvent in vacuo was dissolved either in the K0H-glycerin reagent or in isoocfcane. In Figure 1 is presented the evidence for the state­ ment that ergosterol must be removed before making the final ultraviolet absorption analysis on fat samples that contain this sterol. It will be noticed that if the absorp­ tion spectrum of pure ergosterol ( obtained from Fusaria(38) ) is taken before and after isomerization by the original method, almost 100 $ recovery of the sterol can be accounted for* This means that the sterol is unaffected by the reaction and that its typical absorption curve would be obtained despite such treatment. Therefore, ergosterol, if present in ^ fat containing small quantities of linolenic acid, would

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FIGURE 1 H

O

VU U © O O

©% H H

43 © 09 O O O to •» u o w pH >—»

o O O * o>

o o o •* ©

o o o •» >

o o o % CO

DETECTION

280

i

275 MILLIMICRONS

o

O h3 _

f^OO K W Eh W feoao H O

265

270

IN SPECTRAL OF ERGOSTEROL INTERFERENCE

285

OF LINOLENIC

ACID

o o o «k 02 H

£h

03 i-3 O

S

«mh WEh « CO W W £§8 § P K W0 H

a.W n (-q

•a at 09 -P

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t

■undoubtedly interfere with the spectrophotometric determina­ tion of this acid. To show the validity of the improved method, two sanqples of linseed oil fats, with and without added ergosterol, were taken through the altered isomerization procedure. Ergoster­ ol was completely removed by this method for in both cases a similar curve was obtained. Linseed oil fatty acids were used as reference substances in these determinations to simu­ late conditions qualitatively approaching those prevailing in Fusarium fats. Sterols Total sterol contents were determined colorimetrically (37) using an Evelyn colorimeter, manufactured by the Rubi­ con Company, Philadelphia, Pa. The standard curve was pre­ viously determined (38). Mycelial Weights The mycelial weights were determined by filtration, repeated washing with distilled water, and finally drying in an oven at lj.0oC. for 2i|. hours, or with an electric fan at room teuperature. Hydrogen Ion Concentration All the pH determinations were made using a glass elect­ rode. Riboflavin Content The content of vitamin B2 in the dry undefatted mycel­ ium was measured by a method essentially that of Arnold (2). iThe readings were taken with a Pfaltz and Bauer fluorophoto-^j

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meter* Total Pigment Production The method devised for the determination of total pig­ ment production is based on the absorption peak of lycoper­ sin at 5227QH and the assumption that the greater portion of the chloroform extract consists of lycopersin* The fil­ tered mycelia were dried and ground in the usual manner, and then suspended in \0% HC1 for 2I4.hours. At the end of this time, the acid was decanted, the residue washed with dis­ tilled water until acid-free, and dried at lj.O°C* for 214. hours* A weighed sanple was then extracted in a Soxhlet apparatus with chloroform for 7 days. The extracts, properly diluted, were read in an Evelyn photoelectric colorimeter using a 5201pf( filter. Pure lycopersin was used as the standard (Figure 2). d) SPECTROSCOPIC DETERMINATIONS Ultraviolet and Visible Regions All the absorption spectra reported were determined by using a Beckman quartz photoelectric spectrophotometer. Read­ ings were taken at 2m and at

intervals in the region below 300>t}/#

intervals in the longer wave length region* The

calibration of the wave length scale was checked by the H alpha line of the hydrogen discharge tube at 65 6 .371^ ( 6 ) and also by the method outlined by the Bureau of Standards (125). The solvents used a re noted in the figures. Infrared Region L

The absorption spectra of lycopersin and its diacetate J

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in the infrared region were obtained with a Perkin-Elmer model 12 a infrared spectrometer with a sodium chloride prisi?j the thermoelectric current was recorded on a strip-chart recorder in the manner previously described (30 )♦ Hie sensi­ tivity of the instrument was checked by the determination of a standard sample of ammonia vapor over the range between 800 and 1200

The spectrogfaphic record obtained from the particular strip-chart recorder employed does not have frequency or wave length markings. Suitable calibration points in the various regions were obtained in the following manner: the wave length scales for the A (2530-lj.OOO cm.~^*) and the B (1620-2000 cm.-3.) regions were calibrated in reference to the atmospheric water vapor absorption bands at 37^1 and 3882 cm.~^; in similar manner the atmospheric water vapor

bands at 1700, 1830* and I87 O cm."^ provide calibration points in region B; in the D region the reference frequen­ cies are obtained, by superimposing the absorption bands of acetone vapor at 1230 , 1219 , and 1207 cm.*-1*. The samples were analyzed as Nujol mulls in sodium chloride cells.

* The infrared curves were obtained through the courtesy of Dr* Konrad Dobriner of the Sloan-Kettering Institute for Cancer Research, Hew York. The interpretations were made by Dr. R. Norm$n Jones of the Division of Chemistry, the Rational Research Council, Ottawa, Canada.

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d) X-RAY BIFFRACTION

n

The X-ray powder patterns were obtained with a North American-Philips X^ray diffraction powder camera with 57*33 mm* diameter copper radiation filtered through a nickel filter

The pigment was packed in a glass capillary* The

diagrams were recorded on 35*nm* Kodak No-Screen X-ray Safe­ ty Film, which was developed according to the specifications of the manufacturer*

** The X-ray diffraction patterns were obtained through the courtesy of Dr. 1. Fankuchen of the Polytechnic Institute of Brooklyn, Brooklyn, New York*

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a)

III. EXPERIMENTAL

CONDITIONS FOB GROWTH OP FUSARIUM LYCOPERSICI At the commencement of this investigation considerable

difficulty was encountered in the cultivation of Fusarium lycopersici for the purpose of obtaining sufficient pigment for the study. The fungus had obviously changed its usual growing appearance, and now produced only very small areas of pigment. Considering the previous report of the isolation %of only 7 mgs. of pigment from 600 grams of dried mycelium (8 0 ), continuation of this study under these conditions would have been practically impossible. The effects on the pigmentation of Fusaria by the pH of the medium, by the influence of light, and by the type of nutrient medium employed have been discussed by Smith and Swingle (117) and numerous others (l,7lj.,113 ,ll6 ). Jt is be­ lieved that Fusaria require the presence of certain heavy metals for the normal development of color (72), and Raistrick and coworkers (5 ) provide evidence of an increase in theyields of pigments by Fusarium culmorum when cultivated on a Raulin-Thom medium in preference to other media. Sherbakoff (1 1 5 ) mentions that a medium rich in glucose usually increases the density of the color produced, and this has been confirmed for pigmented Fusarium lycopersici by visual observation (8 9 ). Since it had also been confirmed that pigment formation occurred only on a Raulin-Thom medium (8 9 ) and not on a Czapek-I>ox medium, our efforts were directed

17

towards modification of the Raulin-^liom medium for the aforementioned purpose. In the first experiment 96 flasks were made up contain­ ing Raulin-Thom medium in the pH range from 2 to 9* Both tap and distilled water, with and without the inclusion of 2% agar, were used. No differences were noted between the tap and the distilled water, and only slight pigmentation was found in the acid range. Very little growth and no pig­ ment formation were observed in the alkaline media. The opti­ mum formation of color was found on the acid media contain­ ing agar, but in later experiments colorless mycelia were also formed on solid media of this type. Another attempt to obtain pigmented mycelia involved the addition of 1% Neopeptone to the medium to provide an organic source of nitrogen. Again, increased pigment pro­ duction was not observed* When 0,6 ml, of Vipenta ( this amount contains: vitamin A, 5000 U, S, P, units; B^, 1 mg,; B2 , 1 mg,; C, 50 mgs,; D, 1000 U, S, P, units; niacinamide, 2 mgs,) was incorporated in the medium both with and without added Neopeptone, no effect on the pigment production was discernible. Tables VI and VII also show that little effect on pigment production ie produced by the addition of riboflavin or nicotinic acid to the medium* Thus it was found that variations in trace elements, nitrogen sources, vitamins and pH of the medium did not £ause the formation of suitable amounts of pigment*

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The effects of light and temperature on growth were also investigated* No differences were noted when the mold was grown in the dark or in the presence of light* Almost double the mycelial weight (*7820 g*) was obtained at 27 °C* in contrast to 13°C* (*iiJL37 g«)> but the pigment production remained negligible* Further, shaking on a machine or swirl­ ing the flasks each day did not enhance the pigment produc­ tion* Nothing concluaive could be learned for the results were erratic in all cases* It was found that only beginning again with a fresh isolate of the mold resulted in the desired heavy pigment formation* A similar experience has been reported for Fusarium Martii-phaseoli (1I4.)• In this instance a five-year old laboratory culture was colorless, and had never reverted to a colored form. A reisolation of this culture from the pea­ nut plant did form pigment* A possible explanation for this loss of pigment forming ability, and for the fact that the usual nutrient supple­ ments were ineffective in restoring p igmentation may be the high mutation rate so characteristic of Fusaria (5,118)* This phenomenon occurs so frequently in this genus that in­ vestigators have distinguished three forms of Fusaria: Ankultur, Normkultur, and Abkultur (1)* b)

CONDITIONS FOR GRCOTTH OF FUSARIUM VASINFECTUM

Fusafium vasinfecturn was the subjectof a study similar to that just described. The folowing series of data will jagain show that the formation of pigment is a genetic

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characteristic* Therefore, no clear-cut conclusions can be

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drawn as to the specific effect of any particular growth stimulant. Certain techniques of mold cultivation suggest the use of rotatory drums or the shaking technique (I|JL)« However,we found that shaking of these cultures resulted in much lower mycdlial weights and almost imperceptible pigment formation* In an initial experiment a 5% glueose-Raulin-Thom medi­ um and a 5% glucose-Czapek-Dox medium were used. The results are summarized in Table I. From these data it is clear that Raulin-Thom is superi­ or to Czapek-Dox medium for

thegrowth of this mold. Al­

though pigment contents are

notshown in the table, it was

observed that more color developed on the Raulin-Thom medi­ um. When grown in a Raulin-'^hdm medium with and without the inclusion of glucose, growth was obtained in both cases. However, only in the medium containing glucose was the form­ ation of pigment observed. Thus carbohydrates or carbohyd­ rate metabolic products are required for the formation of pigment by this mold. The effects of incubation at three different tempera­ tures (Raulin-Thom medium, $% glucose, pH 3*4 ) are recorded in Table II. Thus the variations in environment previously found capable of altering the production of color by Fusaria ._ were investigated. They were found to be inadequate for the j

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p TABLE I pH CHANGES ON RAULIN-THOM MEDIUM

Days

0

7

14

21

Myaelial Weight grams

3.0

2.4

2.4

2.3

0.1136

4.3

4.0

7.2

8.9

0.1146

5.1

6.7

8 .0

9.1

0.1331

6 .8

6 .8

8 .0

9.1

0.1279

8 .1

8 .1

8 .8

9.2

0.1296

pH CHANGES ON C2APEK-D0X MEDIUM

Days

L

0

7

14

21

Mycelial Weight grams

3 .0

6.4

6 .6

6.7

0•1081

4.3

6 .1

6.4

6 .6

0.0987

5.1

6.3

6.4

6 .6

0 .1 02 1

6 .8

7.5

7.6

7.6

0.0855

8 .1

7.5

7.9

8 .2

0.0896

21

TABLE II

EFFECT OF TEMPERATURE

7

Bays

Temp*

pH

21

14

Mycelial Weight

pH

Myeellal Weight

PH

grams

grams

grams

Mycelial Weight

13

3*2

*0650

2.5

.2150

2*6

•2281

20

2.7

.4744

2.3

•463A

2 *2

*4255

27

2 .6

.3683

2,3

.3973

2.3

*4646

32

2 *8

•2324

2.3

.3801

2.4

.3911

37

3*4

NOME

L

HOKE

NOHE

J

22

production of heavy pigmentation in both Fusarium lycopersici and Fusarium vasinfectum. Since only fresh isolates resulted in heavy pigment formation, this loss of color must be considered due to mutation* The result of the application of i-inositol as carbon source is unusual* When included in a Raulin-Thom nutrient medium at acid pH, a fully pigmented mycelium was obtained* However, the same carbon source in alkaline Raulin-Thom re­ sulted in a completely unpigmented mycelium* This informa­ tion will be considered later in the experiments on the mechanism of pigment formation* c)

ISOLATION OF LYCOPERSIN

For the purpose of obtaining sufficient quantities of pigment for study, Fusarium lycopersici was grown on a lafge scale in both sterilincubators and 3-liter Fernbacfr flasks* Raulin-Thom medium containing $% glucose was used in all runs. At the end of three weeks, the blue mycelia were filtered, washed, air-dried, and ground in a small laboratory mill. Preliminary extraction experiments indicated that the pigment displayed unusual insolubility, and could not be re­ moved from the mycelium. In Table III are presented the re­ sults of a study to determine a suitable extraction solvent. In all cases 10 grams of the ground mycelia were placed in a Soxhlet thimble, and extracted for three days with the appropriate solvent. The results indicated that only chloro­ form could serve as a satisfactory extraction solvent.

23

Glacial acetic acid was used in other attempts to iso­ late the pigment. Waen the ground mycelia were shaken on a shaking machine for three days at room temperature with this solvent, very little color was picked up by the acid. After heating on a stem-bath for three hours a dark red extract and colorless mycelia resulted. However, removal of the sol­ vent in vacuo left a residue which could not be worked up. Objections to the #se of chloroform as the extraction solvent were based on the observation that the greatest portion of the color remained in the mycelia, despite the fact that thes olvent was already colorless. On allowing these mycelia to stand for several days in chloroform, an additional quantity of pigment could be obtained after further extraction. Evidently the blue form of the pigment was not very soluble in chloroform ( as contrasted to the removal of the salt form of aurofusarin by shaking at room temperature with this solvent (5) )• This condition unneces­ sarily lengthened the time of extraction. Coupled with the poor production of pigment it could have immeasurably in­ creased the difficulties of the problem. However, it was found that by acidifying the mycelia prior to the extraction their color would return to red. This red form could be ex­ tracted with chloroform until all thepigment present was removed. Consequently, the following procedure was devised for the isolation of the pigment* The blue mycelia were filtered, suspended in 10$ HC1, and heated on a steam-bath until theirj

Lm

2k

TABLE I I I

EXTRACTION SOLVENTS

SOLVENT

OBSERVATIONS

water

had a slight blue-violet tint

acetone

had a slight blue-violet tint

ethanol

yellow extract, thai none

ethanol / HC1

yellow extract, then none

ether

colorless extract

petroleum ether

colorless extract

acetonitrile

colorless extract

cellosolve

colorless extract

carbitol

faint red extract

benzene

faint red extract

carbon disulfide

faint red extract

carbon tetrachloride

faint red extract

chloroform

deep red solution

dioxane

deep red extract; could not remove solvent for purification

glacial acetic acid

intense red extract; colorless mycelia; extract soons turns black during the extraction (decomposition)•

L

Jl

25

p color had changed to red* They were filtered, washed with water, and air-dried. After grinding, the mycelia were first extracted with petroleum ether (b.p. lj.0-60°C.) in a large Soxhlet apparatus to remove most of the fats present. The solvent was then changed to chloroform, and the extraction continued for one week. At the end of this time, the deep red extracts were filtered, and the solvent removed from the filtrates on a water-bath. The combined residues were triturated with large quantities of petroleum ether, and

*

finally recrystallized from pyridine. Further quantities of pigment could not be obtained by concentration of the mother liquors from either the triturations or the recrystallizations. Removal of the pyri­ dine in vacuo left a black residue which no longer dissolved in organic solvents, and did not display the typical color changes of lycopersin. During this investigation a continuous study was made of possible solvents for the pigment. This was made neces­ sary by the considerable losses of material resulting from the pyridine recrystallizations and the limitations of the possible reactions which could be carried out on the pigment. When the crude residue was extracted in a small Soxhlet with chloroform, a concentration of pigment sufficient for crystallization could be reached. By this method platelets were obtained, in contrast to the mixture of needl^sand platelets obtained from pyridine recrystallization. Much later it was found that anisole could serve as an excellent L*

j

26 r

solvent for recrystallization, yielding long needles* No de­

'

composition took place with this solvent, as evidenced by the recovery of further quantities of pigment from the mother liquors* Lycopersin was also recrystallized from acetone ( after concentration of the saturated solution) and from glacial acetic acid, but on these occasions a red amorphous powder was obtained. In all samples, the compound begins to darken at 250°C*, finally decomposing at 305°C* In view of the fact that the melting point of lycoper­ sin extends over a wide range and is quite high, the criter­ ion of identical absorption spectra was resorted to for "fc&e proof of purity and homogeneity of the isolated material (131)* The pigments obtained from the following sources proved to have identical absorption spectra ( Figure 3): un­ acidified mycelia of Fusarium lycopersici-2. unacidified mycelia of Fusarium lycopersici R-5-6 (both heavily pigment­ ed and mutated forms), acidified mycelia of R-5-6, and also the mycelia of Fusarium vasinfectum* In addition, the ab sorption spectra of the benzoates prepared from pigments isolated from both the h ue and the acidified red mycelia (Figure 5) were identical. Further proof of the homogeneity of this material were the identical absorption spectra of samples recrystallized from pyridine, chloroform, and anisole. The absorption maxima in the ultraviolet gjegion are located at 266 and 272 )j^(, and in the visible region at £22Wj/l( Figure 3)*

27 r

i

FIGURE 3

LYCOPERSIN (chloroform)

4.60

4.20

4.00

3.80

3.60

3.40

280 l

320

360

400

440

MILLIMICRONS

480

520

28

Chromatography of lycopersin on alumina or Florisil resulted in the formation of a violet area at the top of the column. This color change is associated with salt formation, and it was found impossible to elute the substance from the adsorbents. The pigment was not adsorbed by powdered sugar. The petrol ether filtrates from the triturations were very dark, and obviously contained large amounts of pig­ ments. After concentration, other pigments did not crystal­ lize out. The solvent was removed, and the residue refluxed with alcoholic alkali. This resulted in decomposition and the formation of a black, jelly-like mass. Extraction of the alkaline or the acidified solutions with organic solvents did not remove any colored material. After passing these petrol ether solutions through a column of Florisil, it was found that a large blue-violet zone was formed