ACHROMATIC VARIATIONS
IN
PATHOGENIC FUNGI
Thesis for Degree of M. 5.

Fame: Demetrius Caldis

I925

 

ACEROMATIC VARIATIONS
IN
PATHOGENIC FUNGI.

Panes Demetrius (yldi 3.

Presented to: the Faculty

01' the Michigan Agricul-

tural College as a thesis
in partial fulfillment of
the requirements for the

degree 01' Master of

January 1985. Science.

 

 

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ed art by Ecul'n (1"?) (”4) ', B“afald (l??l) (l7), 3.1.gfiith
(1“”") (“9) :5w3_cfu n“¢, f‘.‘ efi‘uzf (f 7WWIIF{"”EN tal fkwytcrs

Pas been c 'efully stmricd Iy ilcbs (l‘ffi, 1“?“, 1012) (50),
(51), (FT), Lcizixcar (1¢]l) (5?), 00018 (1”13) (T4) and otiers,
tre differert factors, rutriticral and otherwise have been care—
fully varied For the purjcse Cf cbtuiring optinun development.

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C‘xrowiwr 3w crrarism cw culture redia selectec at randcm is nct
conducive to hast rcéults ard recent investisaticns by Ycung and
Bennett (1922) (%1) have shown that the ccnditicns of best growth
and davelcprent may vary wit in wlda lirits wit? differert arra-
highs.

Cofitinunus cultivation of or crrariun on ; jacctc”lar medium

probably not t3? best eultad to it, alac variatinrs in tefibera—

-——~——--“-—_“—H——~—'#-—.—-—

(1) Kurhnrs iv par3“tbesis refer to literattra cited.

ture, moisture, lifbt srd other factors such as depletior of

nutrierts, accuruletieu of netsbclic prcuucts etc. may or may not

e crcise an ir i‘ lu;zce c:. its d€avelcprent as shown by S.e ens and
Hall (19“0) (74), LLQ V riutifi s rt (t‘ r it: (CCCiTiwu ”or
have frequ Iil“ ariszm It has been noticed for sons tlie tbat

different cultures of parasitic fungi in tee berbarium of fichi-
P“n Agricultural Colleée were rivinf off tufts of wbite rycelium.

It was for the purpose of raining an insirht into the cause of

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such variatiors +bat tbe ?rsscrt inves fation was started. Tbat
are these wbite tufts? Are ttey mutants, ~YV‘rthr erial rcdifi—

catic s or bits of sterile myoelium? How do they arise? Are their
characters pernwnert? How do they differ from tbe parent straits?
The time available did not parmit an exhaustive study of all these
questicrs but the little that was learned is rep rttfic here,
toretrer with a reviev of tbe literature available, witr tre hope

that the infcrraticr way be of assistarce to sore other investi—

wator wbo may be interested in tnis Ia sciniting problem.
R} j.» I i ”'3 CF 11113?) L I TI‘JJFJHT 3.7 It: 0

Variations in plants and animals have been ouite frequently
reported. The terms nutaticn, saltaticn, sportits, or variation
are in venerzil u 0. Authors, however, differ in tne definition
of these terrs. Dobell (l?12 (34) defines nututior°ss ”those
heritable modif lC sti ns wbic“ have been induced in various were
in various ricrcoréenisns , while in a subs equeut paper (35) r»
adds: ”The word does not imply anythin: ccrcerning the magnitude
of 0*anpe, its suddenness, or the nsrner of its acquisition. lhe
term denotes a change in gen etic C(Istitution. Inperm nent changes

-—nct hereditary, are modifications.(Uolf—baur)”. Brierley (1030)

 

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(1?) defires rutstic: us "a "enotypic c urge in a pure line",
while according to Almquist (1022) (1) it is "the formation of a H
new constant variety or spec cies 0. lxc ep t hybrids probably all res
species are produced by ruts.ti Lcn.” He further adds that most
autEOrs by mutation do not nean the sudden ar-pme .1% toe of new

constant varieties but simply a sudieu chanre. Jordan (101") (47)

J0

d.stin7uishes three types of Vnfl‘tt Ch:

7

True Variation 1 ututiCn).
Deve lonnert of Let: t ctz racienistics.
fnvi irCr st 31 Iodificatiors.

OJZ‘OI-J

He defines mutations as "charres due to intracellular changes

brourht about by non specific influences and not to direct

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at ptations to particular envirornentsl conditions.” By latent
characteris tic: he neans "dormant qualities in the organism
manifested only in resp on.3e to definite external influences.”

The environrentsl rodificaticrs are "closely ralated to the above

class." They include "the effects of use and disuse" and are

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us
to conditions offered by environment. fiery so-called mutation
are only ervironnent l n cdificatinrs whicn can be lost or re—
tained for a lone tine.
He further stipulates the following requirements which v‘iist

be fulfilled by every true mutation.

l. Appear suddenly without intermediate stages.

2. Be irreversible.

3. Change must tale nlace ir- s

e are deters.
4. Not all the cel is of the paN

l1

stra ns are involved.
'Pfeffer (1905) (61) distinguishes between variations that are

termed indeterminate, sp<3nta " eous, saltatory or single variations

and ttose tbs t are desijzu wt d av gradual, determiru e, and adaptie’

A saltatcry variaticfi involves the sudden aipearance of some new

 

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property in one or few in Ci vidM.l rrd tbis is "... the result

of prevailino corditiors irternsl and external ans it is readily

corvprebensible tie u_;v‘:u.szuz.xl 3C31Ci '..tiC.1'1s ediould f3XClT.E>, 5,, tendency

to saltatory variation and Fence also to corr spondin; ir,ernsl

cbanses. Tbst swltstor? "-"z1irns may be inherited is certain."
s+evens (102?) (73) uses tie term saltstirn to cover varisthm

in an orfsnism wbcse cytolcfical conditions and sexuality are

unknown. ibis is a restriction of the ncsnino of tbe term as used

by Pfeffer.

". de Vries (1900) (97) ir his monumental work on "the Iuts—

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t on Theory" does not make any dis crimirction between the terns
sslteticn, mutation or sport tlfltfl31N31isss irterchsnfjah133 The
Darwinian tern "sinsle varis ti.n" is used to neon tre enme tbinr.
"Sinrle veriaiiCrs are sporadic pbenorera, sppesrins only from
time to tire and suddenly obsnfinr the forms of life. They carnot
be induced at will, but must be waited fCr "(P.31And in snotrsr
place "....fregu¢rtly tbsp concern only a sirrle character an“
tbsn ucrslly consist in tbe lo~s or let ¢3n y of a character a —

ready present."l?.%2) TutstiCns do not recesa3nri 1y appear only

once scoordins to Le Vries, 9.229) on the contrary ”e believes

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in the periodnc rutsbility of species. dn panes Le7-?59 he draws
tbs aw. of mutst on which msy be SET ed up at follows:

q >4 .1‘3 V r . . . 3 ‘—¢’\ .-. ~ ‘~
1. 3:: *7" ‘ 'r‘ spec1es arise suddenly tttbout t‘unsi—

kJ_ “(I (.1 1“th...— S.
2. The? are absrlutely onrgtart,
3. Iost of the re? forms are elementary species and not

vs r3.eties.
4. New elementary species appear in lsroe nurtm e.rs at the
s""e tire or at any rate durinr the sums period.
. 1193 characters have not’irq to do witb irdividrs
voriébility
6. Uutatibns ere indefirite i. e. "av affect all or"sns and
take place in avery concoiv able direction. '

 

 

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7. _. lititljlllf : 3;:‘1’3-3‘grwg’ 1— {gr INK). lootilll o

ire majority of “filfimiFfTWB‘thS for reported 1 iixujteria have

been forcibl induced 'y Cronin; t‘e differ mt :rfurisns on media
to which ncrrnlly injuSrious chemicals have been added. Eobell
(1913) (35) in ti: Chafinition c? mutaticr daiu32H3t take into
ccrsideratirr the "menrer of acquisiticn" and in his paper on
FultathUhi in.Ih;:teria.?1> 33e:(n .\JJ~V l Ijxrzced Cu“ hfx1‘c‘“eonc

‘ . . ‘ . “ ‘ ’l ‘ ‘3 J ‘ “'71 ' ’,‘- -‘ ‘9' 1* A ' 1, . 1-\ ,‘- - » y . a“ . I,
variations discueo;h¢ c y} 1N2:P tho hfiUQIH_E l. PhjSlClOylGdl

and 2. Torpho107ical ru+stiors. Churberlerd and Roux in 1993 (QC)

 

produced an a perO'enous variety of fiecillus anthracis bv cdfiirc

1/2000,Potassiur bichrorate to tie culture flui

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were constant even after pessiné tbe orienisms throuwb animals,

and the virulence of the orrsnism was not diminished. Roux (1900)
(70) obtained similar results by eddinr a swell amount of phenol
to the cultur: mediun std Phiselix (1302) (GB) induced variations

by cultivatirv the orfsrisns at 420 C, or under various abnornel

conditions. This aeporooenous flacillus anthracis was not less

 

virulert tbsn the normal strain sru maintained the asporoferous
characteristic under ordinary conditions of growth.

Phisalix (130?)(GS) thinks that it is the ccnbineu action of
the heat and the air and the slow oxidation of the protoplesm
that produces the arporoyenous strain. Rudimentary spores were
formed in the cells of this strain.

T‘figula (l?97)(57) pramineed an asporozjermus variety of Bacttrik":
_rgmosum by continued cultivation in the presence of phenol While
Villinger (lQOR)(96) developed a permanently hon-mbtlla race of

“CLL‘ s .
B. 0011 aid Wasserzug (199?)(39) a permerently Attila variety of

 

qut. prodigiosns.

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Hanson in 1900 (42) obtained asporogenous races of different
Saccharomyoetes by cultivating then at temperatures at which
growth was still possible but at which spore formation did not
take place. Varieties thus prdduced were constant. By adding
sns11.almunts of antiseptics he caused yeasts to increase their
fermentative power.

Bevis (1911) (66) grew Bacilius'ggli.on peptone broth to
which different quantities of’malachite green had been added
and thereby obtained a variety Which did not possess the usual
characteristic of producing gas from certain sugars. Hod
repeated these experiments the following year with.identicel
results, the lost powers not being restored by sub-culturing.
rho change was accompanied by a change in the appearance of the
colony and the size and shape of the cells.

Almquist in 1922 (1) was able by drying and filtering to
obtain new forms(p1asmodis and microconidia) of'§.§yphosus, which,
however, were not constant. Nevertheless he secured different
strains of Spirillum cholerae_by filtering, culturing and heating
and he also obtained a filterable strain of §,typhosus which he
called §38ntitzphosue. This last strain was kept under
cultivation for eleven years, and it was never found to revert.
It is evident, however, that in this last case as in those pre-
Viously mentioned we are dealing with a sort of natural selection
in which the organisms able to withstand the extreme conditions
live over, manifesting their particular qualities once the over-
shadowing influence of the majority of the ordinary organisms
disappears under the action of the dyd, disinfectant or filter.

This is particularly true since evidence from.single cell

-7-
011113133138 18 wanting, and rather 1'Dub’(;f‘1;_]_ in the early WOTKS. F01"
example,inocu1ations of cultures in the works of Roux, Pbisalix,

1' 1

Chamberland was made by adding a drop of blood from a sneeu whicq

died from anthrax.
Variations induced by using sinilar rethcds have been obtain-

1 ' tlle

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ed with fungi also. Brierley in 1920 (15) reports crit
1 "1 ' Y 7 ' . (" ' ., I) I' .. _ ', 1 e I e
worcs c: AFClChO§KlJ, schiemann anu Laterwun. APOlCflOVSfllJ in

1008 (2) obtained Asp rgillus niger with yellow-brown spores by

 

growing the ordinary forms on Haulin's fluid to wFich 1/1000 f

h
Zn804 was added. Ecgemann in 1912 (72) obtained cultures of

Aspergifius niger «l'b chocolate—brown conidia or colorless turn—
ing to cinnamon colored, by growing the parent culture on media

to which KgCrgO7 bud been added in sw-ll quantities,or tall conic—
dophored forms with Varyine forms of sterigruta, color and growth
by exposing to high tennerutures (40-420 0,). Waterman 1012 (00)
produced different mutations in the way of changes in intensity

of color, difference in nunber of spores and rate of growth by

1

growing Aspergillus ni er ard Penicillium glaucum on Si galactcse,

 

rbamnose or glucose, 15 boric and p—oxybenzoic acid or dichlorc-

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acrylic and salicylic acids. Tie variations were consturt and
true to for» w ether grown from sinjls spores or myoelium.
Brierley (l4) atterpted a repetition of this work hoping to
obtain similar results. As it might have been expected, hove or,
he did not succeed, because, since the results of these, as well
as of all similar investigatiors,depend lICh certain fortuitous
circumstances affecting the organism ferotypically ard cauoirr it
to throw off bernrnent variations, it therefore was not to be
expected that Brierley would be able to so exactly duplicate the

great number of conditions at to produce identical results. fie

reports phenotypic changes affecting the whole culture and which
disappeared as soon as the orwanism was returned to a stards rd
medium. Environment affects different individuals uiiferfiltlv and
unless one desires to doubt the Food faith of these irvestifators
there is ground to believe that, notJi th tending the univer'al

nature and cosmopolitan study on these fu nfi, the particular stray:

11sed by each of thee eirvestirators was constituted genotypically
in. such a ranrer the. t it I"r.:;-%1’)Qlldf,d in its ovm particular way to
different and vs ri able external stiWuli. Schieman (7 Q) discussi:
the ths obtained by her, observes that the e vur7<tiors do

not spree as a respbnse to a ujiivi chemical us3d. A varie tticn

”I

similar to the one observed on cultures where P3 3 ssium bictrc* t-
had been added, appear ed und;r n3rmal conditions on Pulst's mediur
at room tenserature in ore tube but of 173, while on 112 cultures
with the addition of Potassiut biclrc Tite only three rutatinrs
anneared.0ut of 307 cultures under the 1*flusrco cf stimulants

of some sort, a 2.02% cf va riability was observed and in all cases

only a few heads out of the many “hheruS were affected.

E.F.Smith (1300)(°O) rip"rts variatinns ir color, thickness

 

of strcna and presence of perithecia in Neccosncspora vasirfecta

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and Stevencand Hall (1900) (F4) five a series of nddificatirr
due to envircnnent in a rreat list of fungi. irese, however, are
not nerr1rrrt variat‘rls ard are simply what Jordan calls
environmental modifications. Tuch more so since we bave no reccrc
of their nernarercy and the cultures, probably, were not derived
from sinrle spores.

Edgerton (1009) (3?) attempted to ctanve the one racters of

Gloeosporia by yrowirc them for 3 months on Elfving's nutri nt

(13

solution or on potato a"sr tn wricb various substar.es had been

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added e.g. 10? glucose, 1:100,”00 capper sulpn»,e, or a crop of
lactic acid. Frequent transfers on tbe sore kind of reciuu were
rade, but the variations induced in tris way were not permanent.
When, however, the organisms were frown for a oreater length of
tits on the same type of redia variatiors in the amount, nature
and color of the aerial mycelium, color of the substratum, and the

trees vsriat

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presence or absence of postules were observed,

ations did not disappear wit“ t”e eye of tbe colony.
Blakesflrx31h1 1713 (7) ivvhrted nutsiixwua in fucormstnrcflisni—

cal and otber ferns of stimulation. In 92971 colonies be reports

95—30 trpes, the aberrant characters beinj absence, ircresse or

dacrease of zyrospore production, peculiarities in color, density

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in beiobt of sporerriel

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and rapiditybf ny eliel growth, difference

filaments, the almost exclusive production of yeast—like cells

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and the produ<.icn of filuventous nyoelisl growth devoid of
soorargia. Some of tbese are temporary conditions while others

are permanent, tre'oharscters being reproduced for several spore-

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an ial generations. The s.re author r ported in lflfiO (B) a

m

chanre in sexual activity tefillng inward neutrality in both tbs
plus srd minus races of ”ucor rucedo by growinw them for several
renerations at unfavorably high temperatures. Ibis condition is

not pernenent, however, and the sexual activity is regained

Ho
5..)
._

a few generat‘ors of cultivation at low temperatures. He concludes

tbet "tre apparent neutrality rsJ mean merely that we bave not

yet hrnpored to expose these ferns to t7e peculiar ervirorrentrl

conditions necessary.”
The varietiors reported above rave been fererally induced

throuoh exposure to unfavorable enVironmertal conditions. Vari-

at one, however, do rot depend entirely on cbsnges in CHViTCPVQFt’

A

—_LO_

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Imitrifixn: etc. lfllt rqu'be bITfiNJTt sbcmfl: throiyfl‘ irbeiwnfit prwvxxrties

different investirators. Jrsdual adaotive Verls

naturall" fall into tile

0

lass as the factoror returol or arti—

ficial selection is it operation ir one? cases as well as if
those variations ifiduced by t“e use of disirfectnzts, dyes, beet
-

etc.

Rettger and Sherrick in lnll (65) reported several irstagoms
of variatiors obtained by selection of orrnnisms for a certsir
brooerty as e.g. the wcrfi Ff good an (l¢QQ) (79) on L? diphtherize.
He was able to obtain'hyyseno low acid producififi tyres after 36
selections by sirply SFOWlW” tre orfenism on 17 dextrose bouillon
tubes and selectib~ for biéh sod low eoio porcuction. borgurlen

(71) in 1903 was also able to obtain colorless srowtls of Baas.

 

Rettre {r7 {berrick (”5) were able 5, SElect a Whig, :Yf red
strain fror a culture of Fret. prodigiosus Vbic“ was weak iw

I"

obvcnoperioprOperties. ilese Fireire were carried for sixty five
transfers agd tar. fcyrc to be oorstKLt.Tbe“~ Fen ro Kooificstion
or otber crwroe is orVirobrent except the fact of selection. A

similar attempt or a culture of B. ruber belticg§_fsiled. Bucka—

ran and Truex (1910) (17) also failed to establish blob 3rd low

acid p-0ducinfi strains in Streptococcus lacticus. A strorsly

 

cbromoseric straio of B.pyocyare{us was also developed by Retiyer

C)"

3 from a strain wbiob bad almost lost that pro-

I

and Sherrie? (A

perty and had been nevlected for a loos tine bavins been allowed

-11—
to drr down. In tris care we lave e ratvral selecticn of tre tyoom

J
most resistart to dryir" which also happened to be tte one boss ss—
ins the best crrcvrwerio prrrertias.

Coming now to vs iations which could be truly classed es spo.u.

takeous, the WC?“1COf beljerinck19¢7 (6) sic >uld. be first melnii _xei.

He inc} zted from sclizotlcclzror‘Cee sp. uiffe “rt rprslic Cu_;er—

lei",?. iibe :fiiite 2x91 ilk) brvfxn Entrn1'4s wer‘: CCE'bie¢ t, tth t‘rw
iot<_%r*fiea_li-sate showed variatiors. Jorfem‘on in 1’37“? (47) isolstml
L098 Cf )Lsts frcn eirrle spore cultures which s'bowed variatiors

obtainins

l“
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asporogenous type: ofLSaocburoovces pastorishus, 5. elliosoileUs,

 

 

and S. cerevisiae bv pletirr and selection. Lassini (ICU?) (FF

 

found i311Nire cIJ.Ir~V‘ of S. crflfi_rm1tabilis fVuvms that would

 

ferfiert lactose, and rrow red on ero' 8 medium. ibis ob r:Lcter
was permanent. The parent striio was an ever sprrtiny varie y, t“:
variations THWmlfllTT cons ”to t. Kassioi's work was repeated bv

Burch (1°09),-Sauerbeck (1°00), Eerecke (100°), onale~1

I‘m

C\
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and on? otb rs.

Barber ii 1G0? (3) made “Alecti) of it l'.viduals of Escarg—

b

 

' ' r

roflyces aooxslus VHF”LR” in for? one size. as flhU able to cbtoifi

 

- '. —: ~. -. --.»Uu1. - -42 !\. o - g ,. . ‘
varznt_o-s, indeperu~-tly of couolt.vos 0L cvltlvetior. There

cells w*icb adhered in ”rouos and also in tre osrtitl loss of score
genesis. Treso morghclcfical character slice We‘sistefl in a "rest

,' 1 . r 9‘ . I N f V ‘ ‘ ‘ " A ‘- ,r\ ‘ A —-
var'etv of meals. vasir l .1011 c~aractere TuCh so '“jet—r r Jst—

ance to desiéoation eflfi rihj tefioerstures, sod bifher forf“5xfgt:9g

'} f V: ' V ' ,3 1“ - (V V‘ ') V | ['1 -o '. \ _. — . - ‘x‘ - 1" 3 "1‘ 1m “- ' “WI‘I ""
“04-01 w,re ;‘~~tb€9d bk t e res race in mor e deneu uGLFQV t'r‘ *
Aquila-d: .
I s w~ **: «lop m~wuod,vfi‘i‘ ’ coll coomnfilfl oiw’
Fl‘o' t: "C3 T):_l,r‘l:‘.£' t‘ O :1") I” .3 AI 24:5- \‘1\ I '- -1~ - u. " .1
V .-

ii: i be—

8
Elena metbni be selected sin~le Organism aid was able to isola

new races wbicb were characterizei by a tendency to grow into

filaments swd bv less bf metility. Sucb rates were censtsnt. S

milar experiments with fihjvehosus failed to give constant va—

 

- '2 A ‘r_ ”\r' ‘ I \‘c‘ ‘ 1 ."~ ‘ 1 fl '3 -‘ 1 1
“ialxts, tbe same bsir true U; atteMQtS to “JVQlUP aLyurufencup

._-__._.__-

n

bacterial st- .. . e rtrriw cf 3. colt vbicb

p n .i‘ n :3
‘1 x-‘ L.’ ‘ I

 

of fermertiri sucrose and raffiuese :vfl up wwwfircibf Tee. lhe
varietibn fulfilled all tbs require exts firstulyimd b' Him:

intersedi

:1!

1.9. it apnalref suddenly without ate stares, was
irreversible, CTQT}.nF'€‘ {5 appear Ej L:I 1}} E, EVGY‘a l C? arr. 1Ct‘3I‘3, {ii-‘6'. 1'11—
lvolved erlv u pert of RN) 33119 of the parent strain. “

In

rem‘irtt yaru‘ceit f“r ever 500 ger*rntiouso

nun}? Aldfi tbe earliest recorded suflfien vsriaticu wen re

sorted by Edrebtou in 1?CS (sc‘. Tbilv glltijg a culture nf

“flCP18YB he fcurfl tvc fli.stiuct fcrrr, r"? ":7~

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1 I ‘ ' .“ "\ r .1 "l . r- '- . “\,'¢~ .' _.. ~, I ." V‘ -A..‘V
" 7’31 ;" J‘s _ {her} 1 L' obLn‘} CL .hth-LJ L". ..‘~. -‘ 1-...“111’, fhi] G 1511.; VLJul 1;;th fr'l ."

4—.

P,‘ M": . - 1 '. 9 3‘ ‘3 .71 . . ‘ ‘ '3 ‘H ‘ ‘17- '9 u _
prrc gem peritQHCie LL r(Ctles scattareJ ”iii tue meuium. lhe

1‘? p 371"

s. meéstherium. Jordan in r‘(4'7)'.C>bte.113(:i from a single cell

er

3‘
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‘3‘?”11‘ "UL; L a ' p“ LU v tic/81 Lit i .L‘ "L . ' i "Lid? 1 if " "c. l L it
~ . ‘ ‘F w I \ M' ' ' v 1" VI 3 ' A -
A ('- 7 tL' Z. Ii PVT l '1' ‘ 5:519. .19 C~ 1 pl”u_{3 ". Lithb-‘L '1
9* f2? (c bro n by the writ r Fave not previrusly bee? SCOIG,G

arcnc ftrri, but the fern just described seems tn be 019 :1+M(L1

r: the

*4.

- - n . a m. m . .Hv ,-, . a--+
qiuesi;icwl. I“? a, l.itvar 3)»; er“ Cu; ;»lLu3 CLLCL L11:11,. a (LC its:

Sam‘s Glemerella (37) ha believes tbet tbe variation "extiored

J.

above "was really tbs minus4 5 rain of the bitter rot furvus".
He further remarks : ”lbs Glcwerella ferms ebcw sc fucb vari—
ability tbat it is szsible ibmt we have in the cultures tbat

have be en described rarely isolated 'sristiou that may not be

common in firm Asooryczies as a group or ev33 Wibhifl tk3 ferns
Glenerella itself.” phear ard Wood it 1913 (78) ir their mano—
Frafh on t13 qefiue G10“3P3113 meme noti 03 cf tF3 ariubilit: cf
the fSVUS no unarac.=r 33333 to h3 W311 fixed. Appeara1ce of
ryoeliluf is 111313: uii11113. 3LL21;3., 353113, 331_I'12';f(rrr
marxtF3cia, as Ci, 1333“fi\v33, au& 33ra®hvves V1rf 3xtr~mclv i3
1uxflb3r, size, 3‘399: 78? bmrnfi, 1333 CF ?P383"30: ahiiih793:

Cuulfl ’xrt tr303 gyn' r:l»tflxuz'bet—

w3en Lh333 Viriat’ffis LV& 3&vi3fi1v3fit 333 333% to cnflolude "tFEt
Q
o‘ :‘r 1‘ 'C‘ - 7’), “‘“n t.“ 93"“ le‘l"’ 1 n‘,“.:}_ ”
Val“ -rlt .(‘Y -; r'..- . ”3-1 I 1) L_1L./'.1;c)~.. xxx Lb.) o
A 3. ' ,.- _ u. ' .-.
BJPfOff i3 1‘14 (13) r3 ort3d var iflpiflms in Slhwl3 3“¢F9

a 1 ~ -. T n r ‘.-‘ . ' 3. . "':‘ r‘ “‘ '7 ' ' ‘~ ‘ 1’.‘ . . Q
cultures cf Phyccnxoes Liteis In _1.; chirqct rs. urablll in
M -

 

 

1°19 (? 3) not 1031 four distinctly diff3r art t 333 of ?“yllost—

 

iota pyrina wFi13 conuncti3' 31r3 cultuse studies by the pour3d

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in the way of
diffe‘emoes 1G 3013r, prrsehoe of aerial mycalium and abundaroe
anl distribution of nvcnidia. £3 reracdv thésa trnes as possib—
ly elemertsery species in 't' 3 Lex‘riesian {sense or 131.1333 lines
acccriligr t3 Johurwwmmi's usa CJ"U33 term, 13121 3303a;
in 1?13 (2?) h3 3id3 that th333 f3ur st33113 «Ire Similar mi—
sros m1 icallj 3W3 rnfiaifi3i true to type from y333r1ti3n to $37

Ul-

h

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ration. I? u gubsequent rota ifi 1‘14 (30) ha r333rts av

- - ,- . w‘~ ." ‘ a 3 ' ‘ o'. "- ‘ ‘ 4 , ~ '. a 9“ ‘. ~- .~- w , ~ ,— 1
aspfirnzewods 1331tiud 11 t33 3-33 f “338 3311.rumg L3 3 gur3(93tris
a”, r V ' fl 1" “ ‘ " l‘ ‘V '. l' ' . ‘ ‘\ ' f A -\ ‘ V 7-, ~ 9 . ‘0‘ --
I1ish)3w1tuve. L 3 #31: 1 1333? 3930131 stimplat;»u 93331333 3.13

”3w1”3' ary fruitirr b<fi3¢ hut Hot 333r33. Crabill in 1°1F (f1)

a. Q ' ‘ {y ,. . -’ . , ~ '3 3" . ~A 'N,» -,—, . H .1 ‘ c: -, <2 “__- (‘1 ‘
r»::‘.rmt3-3L.; 3.4.33 f d 1. w 13le 1.31.3 1;; 3a. 5.1 r. Jyrium ‘3 v-1" .1313 (1-3003 5.3113719:
”—1“ - -v—c ~- I - ~— - .—_—- —.—-.-- - ,
*3 #3 {+1w ~nn~w~7*-~ +~ \hdlUFV‘ WW7 w; F -J_"i+h t3: Pvrmn 1"““vw
Hv _ VL ”7 L, u ‘L.J./ (J I»; t. - ‘ 3. ,‘ b. 1:: £1 I; 1. \ I I ‘ ~_l ‘LGCN If - \J 1 --J I.) -"'\_/ . LA! 1.1L)

'3331103 tiata W*'3“‘*¥31irvaxf3rr3d t3 tfivarwruis Cn”iitilyrium it

1007. Vutic 2*? 3011330i in 1315 (59) hrwevcr, Cgirwi “ 3»A337

U - ._ -.

 

 

 

 

Ivatirre 7"'Ii;‘t' ".'L }W@*T f\::“ u Jff“ z’t 'L’ b t?“ ‘ “:azi':¢

Ehyllbc+ictz 7??Tfiu "”“le be 111‘?3l’ei bfic;‘"‘ ii if F1r+i“c+—

lv fifTVflflvfifi frrfi;ir“1”§:yvin“!;“fifi J‘. ;%G Cf“3’ir ff 1v ‘ ?“

rv‘ ‘T’l?<a .t fir t bufi,+fftvz “f Vflp ni‘r-‘“®* “‘ i‘ fl”313 fkfe:
I - _. ---.

Cf Q. D"?i“‘ t1““ Wwfbfr. T“ i Gr b711 3 11% ml‘br ‘i~ ‘

wr? fkrcvn Cf? frhfl diffnré t “i'flé SfPrQ a lfh",fi fif fl“

th ”*9” 931,;LLJI c :hhth11 “ virflr P_t vwrv ”Vf ' fi?t"?

in 'thca df“f1"f" “? ‘f‘ ?“" '"*-.13, s,‘:r1d‘¢1;r flilwt";r* : ‘f\ X ,

z ‘ h G)]311)*w tFmt "L?3 1FL4,.:Uf't“ is F Vl‘kft 1: ’w-.5

1"?” ‘gl'w :‘i1'1“)"“" ,tivgb7 “r015". 21‘ r 'rru -im‘ 1 fi ‘

31?? isrlwf9' fi" ‘\ fi' well uh :bvvr.] g7” f““u *%4~x

ylrk f'“ ' f {*1 crfeflisu ;F;"r it .ghq.“3i 9a ~ v rirfi'n:

0* *“tat‘rr. 31th stnaiAs var: LatVOfixlc. utto n‘d “OllaCCi

5

in 1¢QO (RC) fnmxfl
RDG“7R arm
,

"lf II " '.
.— Lvd \.' -~ L‘

P‘r

';¢TVVC?‘V+WA

la in

 

wfirh
17ftlcal £9 CCfilct

u

:33

iuw jyrlnum (v

svwhhyw (55)). Phyllngticta firrififl assuvas

 

viz: sadtate s of

wares
L

on beer wort *3 aid leron Velatir “91+valxzed :itH SO ‘UW
Vqfiircxifra. 2*flrmacchw“13t~> isticc I‘“l*i av Ldnztt i“ £Vfl3VQQV“ f.
F ‘arfitions nw t"‘ “V“: Veiim but AP: 1(Rt Of 0t7°” “9Q a.
Briehley in 1“?0 (14) r~“rrtei a SAUUGD v riqtifin iv a
sifiwle conidium culture of jotrytis oiiggqg. Ike varidt_0n was

in the way of a

2§ black Chan

V I"). ‘

(11.7: 1 :'

stayes,

C

Las,afi far as

rumba? 0f

0010P19

 

ss Peperotium prcducea once alon with

y‘-
K.

-r ,- ~ \ . r- - . " ~- A ~- '
Urp.gf3: cdlturc derived from a elxglc capl-
‘ ' - ‘ 'u—a‘ ‘ u F ~ ” ‘ - O u 0 ‘A ’ .‘ ‘ .
bDPJRP-ed sakddgly, altxout Intervs'1at9
.,‘ ’ ' , , .. . ' .- - -
” t'e hlstor‘ of tfia SLlet v' of LEE 5W~~
(‘hfih a, Tuza heevlcx} start an“;n ~raat
‘ . ' ' A .l q,- . . 1-“ 9 . - «a .
a. .L L: Var LN 1:3 ml} 1 Cu: Dartldule-zr

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character __ that cf tie colflrle“‘
differercas between Durant afd mutant id ot“%P *0P9501051031

,lovlcul c avucterlstlcs. AH exteanlva uni critical

)

JJ'

0?

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ky .c ll

Q

stuiv of tha oricin of tFe unriat‘nn 3&8 XUQQ WLtd tie conclu—
9 a
mien that ttis variation cefihct ho callci a mutation on account

of tta uficertaiwty cf t“e purity of Lt: spacles, twe explain—
ttnn of tta cclcrless form lyirf in ”tFS ShLPBPQlV rare cyance

of a fart

r—Jo

13 catiiiccrcre arisinfi from a cell contarirhted b"

the nuclei or cvtcdlann of a wanotyplcully flifferefit inn

I,

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4 w‘
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Blakaslec in IFSO (3) rctcrtad 83V3P5l varfiat.

, 3:. ,. fl-.- aw ' ‘-\"‘ ,. v- ..."- ' , J-',,
(mJlthma 0f 'ACUI'Y‘vkaT}.SIS RY IJ"‘V$1 “‘t Int- Ln“_b V~«c.facl.l3—

 

(—0-
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1y propagated pure l'was far 10 years. I VLTlL
the color or if t“c cawcactnass of "routh of the mgqelium, in

the lenrth of tfe charanfilum stalks or i“ tte 812% atm abundance

Jo

of the zyfcapures, or more ccfiworly ln the rcducnd 813d of t
cclcxies. Ekcae mutations rewuin ccnatmnt tut eventually they

revert to twe norwal type. Two of them, kCWOVEr, war: JOTS stable

“c‘ ~" 1‘” ' 'F‘?" ‘ . ' u ’1 'r ‘ "'.' .. v". ‘. ".1' ‘ ‘ )- .I ~ ‘ . r‘ I '5 ‘np.
t} 3.. t! 4;: l‘c Qt o x; t; l‘. a. .vutf'f ll“ {*3 1.: ' 52,11 Cajun.» L.L\3\1 le3\. +1.; "1 C‘L-L Ll
- , v“, . - \ . ,,.,, .. 1' ' . n . . - ‘ ‘. ' -.. ' __.
“b"u. ”ad”8 CulLura on war; Iana of naala. LhB‘COthld‘ ere
‘ . ~ ‘ .- ~ An . .- (s na.‘ 0~ ‘ " ‘ ‘ - I‘. '\ J ‘ F" a~"\ - - 1*, “
small, lac {1:1}? scorn alt-i. mt: 2;; rfcspx mas. llze out.“ «Hutu-Ht lac uxi

foOSDOPeS afid 3&3 fPOWfi ft? FEVVW Ian’s thrcuwh 13 non—sexual
vqreratincs but it was cccrsionally fcu‘d to revert.
Dastur in 1°20 (72) retortei variatiCfis similar to those

\. I? 1—. ‘ '70 .‘ ‘ , .l' . ‘\ 2.‘ 7‘
reccrucx Ev Adrertcn (9‘) 1n u carciancm plgfiratum c & Bo
........ w‘

 

(Glnfleralla clj:ulat§_(£tcn) Sp & vafi Sc .). These verfatlccs

 

covslsted in the abscice or prasarce of flarithecia, acarvuli,

setae, ard d_evelepfi:“t of eeriil hyphae, 6rd were fewnd to be

0
. . ' "\ . - '7. *v'r‘ ' r1 .‘. ‘ ‘ . '1- v 1. ‘I“ 11"
germanent. Other vartat wue In tee war cf otafllluJ were fulm
“ r - . n - I - -~ " 1 r‘ -. °
to revert when the cr;ezie m was pegged tnrcgyn its nest. llee

reversion, bcwevar, was ultimately} 1 et. In lGSl Burger (10)

 

renortel several variatlfiis in CclL wt.3tri clu 1 glewujnrielQee.
From sin~le spore cultures, straits differinj 1* c lcr were ob-

talred prorwcinr shnree' A black “1% a white etrel“ was 1901a
0
"These muteticfie resemble the etrnire isolated from natural
. ‘_ »
env1rehieet.
Lee Boner in 1528 ard 1724 (113(12), repcrted an alblro

t&tinn in Brachyeparium trifclil frrm einéle spore culture

.-- -. ~._ ——. O . ‘

 

 

“-

kept arow*27" for three Wears. Ike variatirr started as a write

sector in e netrl uL51 culth
_’ ' _‘ _ w ‘ r r‘ ' . , ‘ a 1
8. NJ Wt .9 C’tlflrl‘ad Biro-Jr)”: E5 "LXtCSM {1011-1) 9X11 l 51,1.C1FCJ. i. .Lk I t b1) J l

lnfle DJQCJS, a.:a;e remalnlny lflentlcal

Q

'0“
Q

U

veietatively ar'

9

'L‘ , PW"; , 1' ‘ ..
the tre pa mthb ed at far t«‘

I-

were reported by V.L.Ct:‘ v.3n3 la 1??? (33
a study of t“c Helw:nt*eefiirlum feet—rot cf wheat he notices
that,ccm sie;ally certain ee ctcrs cf a cclc ry :rcnirN on an

ayar plate differed were cr less from the rest of tte celery.

‘ - .‘ f-r . 1 '. ‘ .' T\ 1‘ ' . n ' 9 I\ a ' ~ "".‘ ‘1" r ‘ - v - - , ~ 3-
and ehafie of CUAlClao lhe Verlante «ere Ebrflrldflt eTen thrvg~

the ccnidia.
A. Blochwltz (10) in 1023 attained from poured gelatin

plates of :qpnrvllluq Vr”°lcfllw- a blue celwred strain whl ich

n..._\-.-”--' -’-‘*”d

 

reme aimed ce-etent T' r over forty remereticee. A studv of the

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L
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F“
(+—
6

r r.
t'c‘xc

(a
t.)

oiwment in the efiecies revelled ttat tie mut t lqcbed color
in the walls of tie coriu ie eri tbe mycelium. lbe variatiofi 5:"
peered only c“ce.

The latest reorrt of sudren variation in fungi ias made by

H. Chaudhuri in lGQ4.(21) A Sultaticu i1 Colle+ .otricrur biolo-

n-‘—-Q-‘ H

 

 

' 11».- '. , ‘ . '1 ,. . ’ ' ‘ ‘ 1 "“ . " I ‘ ‘ c'u . "\
etc.. n.eo. ld describeu as hav1n~ apveareu Once lL d Ltfulh

 

kart urder cultivstiofi'for four mofitbe. ibe eeltation apocered
on an Oat Neal Aoer culture and cocaisted er pink mycelium

l3earin" large sole rotia in concentric r

-.l.

.Jo

-Hfi l-nee while the parent has only lerfe sclerotic. The salt-
ation was constant for over two years on Coors' medium or Cat
Keel afar but it i :vari zibly reverted on beinr rrowo on Potato

rm.

agar. lha saltetioi produces a pink color which turr e reerly

i.‘

black wit“ increase in terp: retuir:.

a _u_ .II. .II..II. .II. .II. .II. .II. .\I. .II. II. .II. .II. .II. My. 51.». an :1. II. :I.. II. II. .II.. II. .II III
I - ' - I I .

E X P E R I I E N T A L W O R K.

The investivatioe we“ started in February T92. lbe followig
fueri whicb were kwown to kive off var'atinue in c lture were
transferred on Clintor's Get Real Adar (23) and Sheer & Wrod'e

Corn feel Arer (VG): Spbaeropeis WalzorurT; k(;x-. pta. Rn 19a,

 

Colletotricbtm li‘Td;‘2T‘Ti_;tbien‘um (bacc.&i'cf_::i.) -r. 8:: Gav. (Ltraiizzrz

 

m

.L .‘ - .
I‘ (a V 1‘ N -1 -.—., 1 ' :1
i D(:ll'.t.. L; Li}! a r) vt‘?_!_"'3 chf 3

kl;

I & 13, are Cledocooriur fulvu: Cke. lb

 

4
“‘ ‘“ ‘w—u

from both tte normal lockinw pcrticxe of tbe culture and from

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Sejtcr ri: apii Br. & Cav. was isolated from dieeuee4_celery le evezs

by the m dified poured ilctt matted to be described later

Wetbode of Iv

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195 of

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micrcs
bad hrep‘q
the tiny pi
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a COPK

“tweftg' is
rot grew.
eitber by be

In

cat ”;/(w1 tlr

of 00110 tctcic-u

-——--Q —- -‘ a o—u-o—fl

as
aticns such

of acfiearanc

tbe cultrres

ctber factors

In the

were used,

orifinators.

lecat‘c“

carefully

lid

cope to

- ‘ . 1
PT-Z.~".:OVHCL

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this

3‘?

pr3pare

I" rv- r- $1
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scorpl

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If! U.

of

U3 liWI

-W-

HC

£Lkl£l

add by

farm 1

wbite tufts

Crrl
“ave

plate

carefully

H. 53

dri.

i

rev

0
in

dilution

Teal

"' I’

‘J (“l

q
E.)

found to

the precese

3”Ut Iliiiun br.

ety

tarp): Ira

. .—
1H

Ml Acer (78), Cat T

.,H,I"I

LTJI}

.
STI‘L‘; U _
L

-‘ (A

A”U

GlTELtLI1

x1 13"1I-1ed lately

used lfl tbggb
.th

P.

t

have

«a
L'

n to

be

tbct rotfiinr zizs but

.lllVTU:

—~—-— --_--

Cf mycelium,

“TF3 1

n

or
..NO cb3micele, cisi.f,ctl t

ture

:Iestiwativna

& Cav.,

Obtain

I

m,‘°d.inrr to dir3ctihns

q

yak»

h" 33
J .
aHHP

epcre I33 pliceu or
tle bifh fcwer of th
*‘ve Si31cle E}WOI“:
1we ‘ t oncé Fctlelg;

been
of t

IN
q ". d‘r‘
«J m l -

r“ .
v46

'3. var"
.J U]

-1Q-

1..

' . . 1
ILK
l‘fiin'r

8'!

3J t
ccrtuwina
ran‘fer.

nifir

, Straits

 

ed.

mum

wwwr
’d

or d

mois

011 A a? (

civen by

watch for

A

l_‘ _
Utrer pee

"5% were
tur9:

ts.
ginf m
the

()7
MJLJ

dot a“

11?"

tlflt CflilgT
plates

(Tilirte

over tbe

tat did

t ed

PM;
L

I
C‘. A ..\l

I &

LS: {Itfria a-i i. Dr.
The plar was to

vari—

II

I
(‘5‘
L)

uliurity

at or

Odin

ir

Lifcc

- '- , A A - 3 ~ v " .E: ~.~ N ‘. ' I Av~ ' T‘ . A}:-
Prune JULCB Acur PTJPRPSU L3 t t V~ ’ 3*V9-?er<e ts Co of UStrblt
‘ I ‘ _ . -—‘ .~ ”-1 -. .- '7 A 5‘ , ,~' '1 ' w - in ‘ 1“ . -n. a"
Ilch. (28), Jdtrl9ut drctn Aqar (we), vaQm bQHLWetlc Aver (94)

R1 rda' efntVetic e~ar (E7): Lecfiian byrtVQth Afar, the latter
be Mln_ essentially Cncns' synttetlc 8T9? with E tires e3 finch
3/5 Aspararlu per 00 c.c. of medium, Potato and Carrot Plugs,
Corn Heal in 10000. Erlagneyer flasks (7F) prépgrei by adlit"
a 15F7”C01fll pe” fluek wit: 4 times as mucb ‘rter, autoclavinfi
at 20 pounds of pressure for 15 mlhutes aid fifially rice flasks
prepared in tfle same way.

llmaCNWDTicels used werfifixduxb'e Analyzed”, 51W} the filSSSfiare

«as either Pyrex ur Jo a. All glass culture dienee were carefully

cleaned by imtersing (ver? 1*ht in cleanin: solution, followed

,;
,...a

by fcur rinsinvs in tap water and (he rifisihfi ? Ht; ?_°s
water. lbe water ueed was dietillefi Hater wlicb bad beer reciefii
stilled in a block-tin still or in special cases (PH experimente)
in a Jena Glass still.v

APPEAR NOE-3 CF ‘1::RT..TIUBS.T1:Q IA.‘3'I.’);.T::».I‘C.Z-13.; of vegr‘AAtlc‘is w‘l]-

“Q -I -‘u

 

.1! w.Cn~ DC- ' .. .. .- n.
be LLSCUSbUh _cr much f‘n”un :epdrately.

.1 var. acil Br. % Cav. was isolated from

 

t causes leaf end stalk spotting,

O
H
[D
"D
5:.
g
0‘
,..J
’3
"i
g A
A)
5-4
J
( f
.4
3
CD
*5
(D
H.

, 0?”, . ..-
the algeaee beivc khown as Late Blight‘Colery. Beach 13 Lew fork
(E), Rovers in fillifcrnia (1911) (69), Coors and Levin

1i Tichlran (1016) (97) and eeVerul others Pave wcrked with tbit

of tbls furcds cw d1ff?“3flt wedla. Six slurle strra ieolaticns
were made on Cat feal Are and trawsferred on Corn Teal A :er,
Prure Jutce A.er, Benntut Syfltbetlc A”ur, carrot Plug aid Corn

-- -./

Heal Flask. lbe orvarlu frewncrnelly on all tbeee mslia far

a week forminf a black d3nse growth with nurercus pycnidia
scattered over the surface of tie wedium. lvn days after irccu-
lation a write tuft was ft 1C i3 strain LO 5 on Corn ”331 blush,
forming a strikini contrast with tbe normal black fircwth. (Plate
I, Fig. 1&9 Attempts werw fade to transfer this white mycelium
to Richards Preparaticn Dish, Oat Qesl Afar, sin Jere Teal Flask
but it inv ri bly srefljet black in the'n3w culture.

The same strain cafe 3 second wbite tuft in a culture on

Prur e Juic Adar which was tra3sferred freq a Leoniar Liar lube.
The white tuft was trcrs -fe ed back to Lecriaw medium, but it

grew black axain. Strains 1 and 2 bebsved in a similar way.

Strain 1 tbrew white veriatiens’on Oat ”eel Afisr and Leonian

‘1 K‘"

Agar but both teese reverted w?en isolation was attempted. 01—
n

milsr \sristlvns of stalin 2 on Cat gesl A_ er a d Lecnisasear
reverted on beinv trsrsferred.

13V

SDbaeropsis WALlcrifl 9K was trausferred from tbs Iich. Agr.

L ‘

0011- Experit'-1v3='1t Stati 1 stoc k c Altures ere it was known to
1. r1 . . ‘ '1 0 ~..
ber ave eratit ally. ilere was soma aifiiculty exp 3r'e.riced in
a
cbtaininwasinsle spore culture of this fungus since it would
not fruit on Corn Real Afar or Cat fieal Afar. But on being grown
I in a O
on Coons' Synthetic LiquifiAprcpsratLen dish it fruited aburdartly
anfl two sindle spore strains were se cu13d frcw tbs diluticn
plates. The. ese strains were grown on Oat Iesl afar, Prune Juice
Afar, Corn Teal Aver, Cert feel Wlasks and Rice Flasks. lbs
organism grew luxuriantlyIprcducing an abundant aerial growth
of a greenfilt blue green tinge. The mycelium at first we 8 cottony
white but later tUPIBd dark or evem :HeKrlv black.

White tufts ache ared 01 Sara zeal Agar but on being trans—

ferred to Lecriaw Agar "2V3 ri.se to a dark gray 3 owth with

several black Pycridia. lbe strain was then discarded. -asler

(43) in 1013, 1y aescri

‘ - ~~ v n ’ " . ‘ ‘3" ‘ v -‘-l . l‘ r “ 1 'Y -‘ . ‘\ .-~1 I‘ r. ' .
Black rot f1n_us on different ncuia, 3 3s: colonies are at«fit

first cottony, after 2-5 Cuff tb3 subserfed threaas a 3 green r
blue green for about a week, after whic‘ tie :r(wtb i: flark
or even black, but almost without exception the outer ends of

the aerial thread maintn‘n th3ir arisinal cottony appearance."

I
q

Brooks and 33 Ierritt in 1013 (16) describe tbree strains of

\J

' fered markedly 'n sbate of

1.4
a "<
C)
"S
g
“.3
F?
3
J.
‘J
3‘
..J
H)

Spaeropsisjy3g‘__._w_fi
pycuidia and spores as well as in treir pathog3nicity. Ehey do
not mention, however, any other cultural characteristics. Scott
and Borer in 1908 (73) anu the numerous other investirators

who have worked witb trio funjus do not describe the cultural
cbaract3ristics. Clinton (22) attributes the black color of tto

rot of apples to tle mycelium of the fuufius "which while at first

hvaline eventually becowes dark olive."

Colletotricbum lindomuiiianuu (Sacc. a Tarn.) Br. 3 Gav.

—_._- w - “nu-M a“. man-W --.«

 

 

no... fl”...— .4.

 

Expo Sta. Strai3“£., was also trarsferred from tb3 I.A.C. Expo
Sta. stock cultures. Eilution plates were poured and with the
precautions d3scribed, two oinrl3 spore isolations were made.
lbey wer3 fro n on Corn Zeal Acar, Corr 3,31 Flask; Oat £301

A ar, Leorian Afar, Prune Fuice Afiar, Rice Flasks, Potato and
Carrot Pluvs and in all cases the organism orewnormally. The
mycelium was colorless at first but it soon turned black amw
the pink spore masses were scattered in small pustules31 mm. in

diareter,ea the surface 6? the myselium,whieh grew within the

substratum: are follozinr are short notes on the apjoarauce cf

 

1‘l'.|‘1il4|\11|l[ Ill 3

this fuofus co tbe fiiffcrsrt reiis Used . Ebere were no differ-

ences between the two sirmle sooré cultures fr3:n Strait I.

f1

Lecnian edium. ibin colorless mycelial rrcwtb. “c3rvuli ard
pycnidia. No discoloration of subntr tum.

Prure Juice soar. 1b‘r ht blac " erth, CC"Ori“C ertire surface
of 11.13. Blacks ed substratu.3. Ace rvuli scaIr .

00k Frovth COVt'BriT" e‘tiza

,-

011+; H88.]. 11.9511“. .-..b l" ”1870+. bl Sill
rt. Bla chred s1betratur1mrtirely covec3d ?{1th
acervuli.

3

Rice Flas CS. Abundant black trowth coverird the surface oi We—
dium anfi extendin* dee, irto t‘e substratum. leep rnuse
”ray and dark olive Fray aerial frowtr Hit“ scarty Jlite
tufts. No spor3 1asses.

Corn Heal Flasks. surf'a33 covered wit? black growtj. Rhite tuftS°
Acervuli verv abunnrrt- Ho aerial ' 1

Carrot Diur. black r'rr‘.'.’t‘r‘I coveriro enti~3 surface. No score
pUstules.

'ibe Characte hri sti s of ”rowtb as Fives above C(TT are wall

ibei b" Beach (4) in l?“3, Scribne r (74) 1°Q

’1

ritb those des
and otbers.

White tufts appeared no tbe Corn Zeal Flask arc Cat Teal
[we sla‘fits t'r'tbiz'; 20 days after roculation but transfers fro"..-
trese on Corr Real Flasks azi Uat Jeal near slants rrew perfect—
ly black. Other attempts to be describeo later (p.36) also

failed-to establish a abita stra ir.

ColM ototricbum 11o”*31+r1v“um (taco. _&_:f:u.) br. & Gav.

 

 

 

Exp. Sta. Strain II. ibis strain was known to be more persistert

M Q - - a—A-m‘

 

in variarility. A transfer from the Exp. bta. stock culture was
. 1 . 3 3 U- M , , in a _

made on corn .eal nin a1u or c‘oils solutionhprepurat1Cn dish.

30 spores were fourd on tbs secnnd firm 1: Cilutior plutr s treat

difficulty was experience" 13 weaiufi sifiele s»ore isolations OT

“‘9'." TILL ()3. l.| 1“ leQ‘

A J

"5

”POUR 0% d ff? -Ht sets of WeCiu r3 cescribec for strait I.
SM ' ‘ ' ‘. o .

lhe Frowth VI8.! trynmr} 1r botr StPLIYS an; tIe cescript one

viven of tie rrowth of strain I, CL .53 differert rgdie, erfly

equally well to stroi? II. For more white tufts appguIQQ,

however, especi All.“ or. Cor}. -.e:;.l Flask arid L«at “eel £15.?er (-1.331

H.
0’}

these Variatiors proved to be crtstaht. ire folloni:'
accourt of their eye ararce. Cirrle spore culture no 5 on Core
Keel Flaek Drofluce: wbite tufts Cf fiycelium 20 Cars after iro—
culetionl A little of the white m"CrAium was trek sferred on

"‘

Juice A ar, corn Teal Afar,

(D

Oat Teal Afar, Leoxian Lear, Prun
Cornfieal Fla '—‘ ' 2rd fro.r1 t7‘eee fifteen subcultxfl‘es were rattle
throu"hout the sumrer oo 3 veriety of redia, the furrus growiro
‘3? cct]-y white aid ferric? a rather correct rat g7‘* very little
aerial nycelium or the aurfrce of tre Corn feel or a retrer slimy
layer on tie surface of Out 'eal Afar alerts. lbs strair was

'never seen to revert. Plate I. (111". 1435‘ :fi‘o o..rs th OlltlT'B'?

1'!

.J

of ollet< tr icrum .‘Li.‘-""I—‘=I-:1.1t*:7:_:'-‘*I'I- -319. Ste. ctr. II, rigiasrtows
e. IlltUTTelfiivlif* thrwnv: off xfieite i;uf.e ineile f“- .flflhsrw:-rq a
corral culture. Plate Ill, fie. 1 sknwe a CLlLUP? of the wiite
fcrfl.

Sirrle score culture Ho 5 trrew off a ti i:2.l r V1.1"5atloi;

on Prune Juice Afar which proved also to grow normelly, through
several rereretions on: or different media. It rover reverted.
ire otber sirrle shore cultures were rot seen to throw off such

variat'ors.

Cladosporium fulvuq_0ke . Four eiuele spore cultures we e

 

Secured through dilutior plates poured from a transfer of tr'e

/’

 

stock cultu“e of try

orflsnism free the ixyeri Le
funrus causes a leaf mold of tomatces “Cd it was stuowed

ely bv Yskemsnn (F4) in 1918 in *tis labordtcry. I: “a1
paprr no mention is made of any variations uppearirg ix

production

f“d *1”LIS 11' s 1 tea.. ~r:cgr

of velvety olive PrJe
She 1" our 55 i "ifl f;

eal FIHSCB and is ab

white onlony arisinr
ordinary olive—qreen

1
(Rate I, Fig. 13)but
‘Cb

colorless hy

I

cl
pkeeACorn

of

to Frow into

cclor.

spore cultures

1

out 33 days

I V ‘
frvfin the

\

colonies. A

Fe

m purple CCICQMCE

smal 1 Gus?“

ELPQlN
ceflter of.
pic

atterpt at trat time to

\

CPITtle.F

ion like

A,
\J-

were transferred on
IR). 4 sftnfiml a s:
a, thick mess Of

ttuve of tfix3 Fleflflc we

transfer

'3 1

CW

it‘ll)

Corn

small

8 taCe

‘7‘
.I

be

..e(,.__ 8.1“

‘5

failed pre—

'Jiti write

sumably because brown spores were taken alone
rycelium. Wren, 1MWoven, tlo Write u; p‘u e war.

again ir

Lennian Afar, Corn

grown since for two ye

1"

ras never reverted. lee

mentioned hove: Coors o

8,

F

ma.

Flask, pberrgi leaf

at
t.

fair. ’

it we: possible

parent streinaon P

Afar ard Oat ical qu
are (I u jraet variety

list
Corn

JutIBIiO Af&r,

Nutrient Broth he

Synthetic Aver, a“d Lime Bear Inf.1siou Afar.
Besides .Mtril: F0 4 the otrfr strains th

at different tires avd if Prey oese successfu

effected. Tahle I pres Hts in account (:f U e

variatiros ie t“3

used. The different

cultures are idehtieal e

d i f f e r r t

Whit-

strair t}

.3
v

forrs from tt

{3
I

rd rave been used in

UP.

jeal

E8

allowed t

3
J

J-
\J

to obtain
rune Juice

of‘f“eéiie

er“
:L;‘

o rrow

re write

Aislu

it

of media includes besides ties

Flask; Rice
er, Czepeck
rew off white for“m
1 isolat’ons were
rooearwroe of toese

I, . ‘. n . ‘Vu
LLllI€3PSIJt

different sinfle

the various

redid

'r-x
S ' 9

Cl“

Q‘.'T‘.O
V4» .‘-' V

ri—

a

 

 

 

 

 

 

 

TABLE I. the adfvxracce of aohreretic varieti rs in four
sinsle Spore trains of Cludoeporium fulvum eke
ot? rwise id rtical, on a series of stdidnrd
reedia IPadh'vsacxzses “pr“gxiz rn,lkxnitive ——.

F-————-————c-———qp ————— ——-—I-I-—— ——————— F——--.- ————— r————————-q|—_——_
KHDIA o StreinI e StrainIIDStrsinIIIeStrsin IVQ

‘—-———-—-————-—_-Jr_——-¢——‘--——P——-_~-¢-—— r_‘—-‘fl-‘-JI__—-____-qI—I~-_-

Corn Teal Arsr w w a 0 as d

_——-———-_———-———4n———-————~—Jh——I4———o-——1P—c—¢—o—————¢-‘Jr—‘n—da—‘d-‘un-n-u—u-w—

‘ 1

Corn ?eal Flask? -- 0 -— & M8 d - #3 l

-—-—-—---—---——Ip————————c-«ll—I‘--~—-_—-fiI———————~-‘r--—‘_-——-db

Rice Flask o -— M -— O —- d -- ¢

- ———————— --—-‘—-q+«I-n—u—u-u-n—o-u—Jr—n—a—n—n—c—nn—u-nTp—_——-¢—-.-—-1P——q—d-—o——--—1r

Prune Juice Ag.0 —— 0 —— m —— 0 -— 0

-_—_———-—-————--1 ---------"F——- ————— u—Jp ————————— 1———- ————— +

-- I .

Oat eel seer c M! c MM q “N 6 —-

a.--_____F—.:.—c—n-ndI—n—u-I—un—I-o—a—I—i—n—a—u—urn-I—d—--.-4-———---—¥-—— ———————

1.1903131.” Act-“LI" C ""'- " “u" Cb -‘7--"- C -- A) I

—--—-—ud__----_d_'—— ----------------- -—- ------- —-- —————— q.

Coons Aver C 0 0 an 0

-_-_____.-—-——.-——— ——————— .‘u——— ————— — —————— —-———_— n- _____ ‘r

Richards Liquidi —— h m &

________F————l———1——I-—————— -4 —————— --—-l —- ——————— Tau— __ _____ d.

Carrgt ?lufi e -- e -- 0 -— -- 0

Potato Plur

--—-____ —————— -—1— ———————— -I —————— —---d—-———-I-—-o—— ——-—-—————-J

Nutrient hear 0 0 es 0

l----————---—---J —————————— .:.-n ———————— L——-——-—-——-- ——-—-—————:L

 

 

 

h~_.—____fi_-—,_.._u——.—q_—-
“——

-.—_~___—.—_m~—-H._whl

—.~‘-_-...__.—,

—_._____——v.——.~_ri—-

 

-— -_

bl —. » __. —-.. 74

— H

-- - _ 7— * ~ -

— _.—

O. —_ - _, , ._
._ o .. _ _
-_ H v .—- -- .—
_* _.

.— _. fl , ...l - l
_ -_ .— h -. —.

H—pw

.—

Hhkhh~‘~_¢..——.

——-.———~-——

"Hwfifihk__fi_l

_._.—.77 m;‘-—.‘——- -_..-_.__—..-- ._.—.__

___...C._.~..—..._..._‘._V_-Hw~mfi__~_—_H—~____hn.fi—..._r

 

 

 

._.-.. .7“.—~_~~—————-—~.—_‘-.—-—~——_— _..—..—.
u——. o...—
—~ ~lhhn—~_¢—___———__ *~~.f———.——-—_——.__HC—.”-n_—__~— .—a—_—_-—— .—..—..—
I .

.— -—...-‘ F—
_._.._..,-_~7*-b_~¥fih———fi~_h~——.———h—»-——‘—__—.~.——.—.—-« _____—_._._. ...__r ._..A .- ._A._—..__....
M— —_‘—-_ —— . ‘ .
~-_g._.._k— .* _—h~~—h—____~—mF——ck—.—_-_,-»_.-k-. , --—,._.- H-_--

.
l
__.v _. _. ———-- —-_~.—_—-——.w-_~-_.—_-.-—-— ._7k .—————.—.._-———._..._.._.._.___—.Mw—__I ‘ _ - -.—_
—..—_ .._.__
__-, ,. - - ,_ 74—v r r.- —“ ~_~_-—7 v 7.7. -H-7. ,-_._._.__
i. —-—- —-————~———_
7 .
— . _ ~——v. .o. » —-‘—_- ~“’—_»—- -uhi -7 ._‘-_L.-,-_...._
_..-_ _ _‘.__—.——..
._..—. -
*__._.._.—_—_. .-_‘__.._H-—._..—-r..—.»_-—..- ”.77 - . +w-~-—-——””—A-——HH_-_ _____
w— —— “A
~.__.,. —._7--*——‘-—-—w«-—~-—-—- —— v— - - «r-—P—< 7‘- .. <7 ——-— __.._....-- m-.._fi___.___.___
__ _‘__._._.fi_ _.._-.— .....C VlfiM—-—C—r ——..—--———--——.. _.__._-_»-_—.a—.—_.vv-

 

 

 

 

 

O
f A
u- —
I
r‘QIT‘. u- p‘ , ‘v I ‘1 7 O Q (— .~7- V7.» ‘ ‘ \Or‘fl ’-"‘! CV- ..--, r't-l.,r7-- “Ta-T ‘7Y1«g '7'?
C - O... .I-‘ A \ JP. '._ ¢\ -.L. -.s " ‘11..r.-'.' J. \‘r! A. -r[~ «I '.~.."V- EKJ‘I\, c .-.
A- -.. _—-~ .—— - ———.-——.—— - - — o-_ *.-—m—.—- —.-.I .....-._ _——-_- .u—u. ”fl-.-
0
‘-_w’ .. o g - "~-, _‘ 1 («fl _o ”(‘4 t I." h..."
',_3 grewvf‘ 1- 3"“:7 (r.~_) , .A' ‘ "“ ‘ 3 :’ ’- -‘,;,..\,C,"_.)’,
l - --~ 1‘ " 3 17“ ‘- 17‘ 4‘ ‘1 1" .- ‘ I
IL; P :‘t " tk ‘ .... :J :1- . r 1.. . ‘J ' ' |_‘ r _ ' . .4 L) (I t .
-—-—- -- - .— “*H b ‘ —.o— ~--—fl

*— N“, 1-: m (Vt-m: -, , r . :‘-°.. "w. we. - 1,. ' a ,
‘1 'u( " _ )114 ., . J _ d‘ - ‘ 1 1L) ( ‘ C. ‘ L L\.-' ,CU. K d. ._ - ii I‘ L1.- ku -. k ' a
l“, I 03'} : ‘f,: +, " (': " T Ci _ 1' ‘~ ‘3'le H a C t-“ C"'-~ J "T C,

t““ ahrva we +ivwvr ~i+?'“s 5rd W~P f“fl?fi t. Cirress‘if ¢"'”i—

u
. . . - ,— § u. n..- A yak/U

H
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fi
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e
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u.
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:1
Dr
H
3
I-.

< q p y : j .2 _‘ _ V I O . A ‘
4 » ~ , ‘ - ‘ a - I) w .
b .51., VT L) r‘_.‘ (‘4. + l ‘ W.\..CQ' 1‘ -1; I, (‘1 1 L _ I- I a 1' ~ .. It: ('.‘I \ C 1-: -L\ ._-uf‘ L \

r‘ 1 "I ‘L' I " er— 5-1: I’ 1- “ I I l‘ I I ‘7’: "r “‘ ‘ v“: - F“.“f )Y‘ q J t”"

L4 .L J ‘ — l I 4 1L . un— - . l —» . I
fi~
.
w " o ‘
fur/‘1'.“ ' l (\1‘1+1‘T~)r\ 1" C" 12."l_' ‘ at L - -- ‘ +1": "‘ i "fi"“l_ +' F'-
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Clmllar r0}&v10hr as to Chin? pron cu or by Hgorntrlcluw fame

 

 

Mhi“. fuhruq Evcdquu a brith Tufi cnlcr whauaver t¥e hyvhho

.L

chme 111 ccrflwaot :thh an; aI'TIBBt, 'turfixip, 1v'nuirz, rflnaat, lnrwaxl,

2

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J-

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U'

raoia r103 1n unfurs arc peows no a &ffected hy plyeéeai
factorfi Sufih as acidity etc.
1a%1e II pvucaafs & co 1brativg desosipticn of th yrowt?

media erplcyed OUrinf the investifiutiun.

CL '1, 3. . *, .,,.‘.- \- ."A .,,' ‘4?! ._ -p
u CWWCTLL (n1) V‘P“ u:2» fr: kaCJl’uJCL L; 0F7023.

TABL; II.

*““"**’*'1

Medium ‘

Corn Veal .
Afar.

COT}: ‘13:,21
F1" {gm-Lo

yr
r2+ rv.
’Clv .-“A(A
.‘

aft? 0

---————--—d

Prune J nice
Afar.

—----_—-_—q

 

7V

__.._..-_.___._.._.__._---_.._...i

 

 

‘————D——-—-_——_—_—~——————-_

p———--———_———-—-

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, ,
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11“-. a, “1
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plainly Lushy Lull

?hores.

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rv-‘y'lh 21",; 1‘

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rcwth.

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O

 

 

 

A...
l---._—__._ , _. ., ,, - ___________‘.;_....é_4-.»_-__ _- _- r >— VA
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Table

"""""'"""'1

II

Medium 1

-—_—-—-———d

Nutri'
BPCLHI
Agar.

----.——---q

Rice Fla?

-on—u-un-I—un—i

Carrot'
P1132.

Coons'

SyKLLetio
Agar.

Lecnian

’- 0 0J— ' .
LyhubBLLC

fi

Agar.

q
(J\

k

b-———-_——‘————-—

-4

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LP crlcr. vastratun dark.
b-————-—--——-—————u—uuo-u—u—u—u—u—a-I—JD-n—u—u————————————u—-—w—————q——du

Tedlum FFOL "th. White. .
Ldbmfilgwd *rcwth citrino
or faiwt Dbrdwau: Bo
Qjfirbs.

 

 

 

l-

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.._...._.._.. b__......-- --....___........_.._..__.._..___.._..___..-......_.._.__......._........__+

Czane,k SW“fflCQ rrcwth dc*n olive arc;th alurdcnt, white or
“OXtPCLQ ”ray, priptirr: f"t9, ViKCQCC?S f?%f afifl liflft
Afar. ficrk (live wrcy 0v liNhf vinacwcus fawn. In tn?
‘ ”5????PVS féwz. LU: “idiuw meiiuw a Lerk Kalictrope
C’lCr~q at“? bulic rcfie or ”Fey.
"1 accc"s '30“. '

-----———db--———-————————————————————_r-—_———‘—————-o—u——--——_l—————H.

Lkivc's C”rfaca ”PCTth huff: Ci— ' White 7L6 honey gellcw
Beet trirc, diflj.c3iirire ad; tihfe. hlflastrutufx4nassiuw
LV"n1-1'3ti43p13,3f3 C. 0 "Ph if”? fllf t3 0 ._.1\-'_b"‘ Llfi('-‘l."_:':.

Acar. cfivéffir ““me brcxn.

_——----——Jp—————————---—-——~-——————————_———-—————————————————-——-———d|

". "'7' 1‘ a -‘ . A ’1' . -_J— .
DJ M lulljfl} ".' '41}. ".51‘1 tb o p-L‘b SJ! ljlfifith
Bewfl 'Thltbé'bufts. Lfifl“8trafllf Berixj'brcwn:
Arar. ollvucecus black.

-—-——‘-—Jh-—-——~——--‘--—_—-——‘___-——H h———--— ——_————————-——~_fl_—‘,_

.vr ('1.~ ' an rpm r+‘r\ ‘4/'1 n- ' '. dxr L
j_"l:‘)_‘ W"? '- L. 3 t _ P“ U 9 -.‘.,’l,|"'}' "g'r' .

1° 3}“. 1'.li).13"'€:&.1'ill "I‘ww'fl'm very pale
,t9 0. olive 1? color. Ali“h+l* licht filtkisF lilac in
purple or wfiite hyvkaa «17— cclsr. I
Ed with tFG brcw;is% Olive
crowtfi.

L_______l________-_________-________L_-_-_-___-____-___________l_

m fir l, 1 r. - ’ ~ \YI".
lofidto Acnvc(rt rrcmtb, hpohiu
F "6

 

 

 

 

Ix etngdg' of‘ tlt? BTN‘Vfi‘ tafiilea S'MJLI3 t}£it lmufjw p grazfig Lgul
variant var‘fqiiead cc>v“'sra"=_f..=:"ably in color zine-:3": {grovm or: diffcgcrm
media butthe variatins were along parallel l.nes. Ihe parant

form varfled clxnwfcallvc—tawny gnaccs .gnd ,'bhe varlalt 1n

" I o 1 0 " .~- .. "‘ ‘4 . .' . ‘ _ .n, , ._ . . _,. . -.- - .. .
11"}31-tg3—p1fi .:.—1 1130 f‘, ”Aid“ 5 . .L} t: ‘,73,,I‘_'_&7 ‘5‘, new”! 001 1:511th tbl‘oyil'f}:(xllt,
v . J \ r . .3 ‘. ~ r . w. ’ “- 1’\ ‘. - -
Wkllc t‘v gt? 1» Cr-tLLu m ~T”I%JJN3 qf’uu «ff t.) verlnrt fcrr

i- ‘ ,‘ m ,. - r. -— }\ . ‘ 3‘ {w I ,‘\ -~ V‘ 1 7 — . .
nqa and lcmato ctpms. ,c SUOPdv Mar: yrcuucec by the varlprt

at any t‘me n all t

—. N
v.
._ _.._‘_..___.__________._..._M~.———.——»--~-———-——-r --——-H-——————~c--o—-—<—«-4--——~—————-————————.———-v-—~—_.—_.____...
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t
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,_.—.~._.._.—~._~—w-—n—-—-‘

 

hw—‘F—l——_y—_—---——a—v——ah—~, _._._

 

 

v
._ u I

‘P'

 

 

‘r

-M

-51-

line Bean Agar and Nutrient Agar the surface color is rather
gray while the color diffusing through the medium is nearly
black.

The variations in shade of color on the different media were
not constant but were lost on transferring to another medium and
the color characteristic on that second medium assumed. Both
the parent and the variant were grown on a set each of Cat Meal
Agar, Lime Bean Agar and Nutrient Agar slants, the surface color
was noted and then transferred to Nutrient agar. While the color
on the three media referred to was variable, there was no
difference at all when the transfers on Nutrient.agar were com-
pared. Table III illustrates this point.

Table III. Variations in shade of color are not constant but

characteristic of the medium: Tests with Cladosggrium
fulvum parent strain and Variant strain.

 

.——-----—--.---------------------------—--—---------c—-—--——---——---—

Medium Surface color _Color on being transferred
- on Nutrient Broth agar.

Parent
Oat meal Agar. Cinnamon Brown
Lime Bean Agar. Light vinaceous Citrine Drab or Hathi

lilac Grey at places.
Nutrient Agar. TaUpe Brown
Variant.
Oat Meal Agar. White §_Light brown-

ish drab.
Lima dean Agar. " " " & pale vinaceous White or pallid
fawn. purplish grey.

Nutrient Agar. " " " & Pallis vinao-

eons drab.

 

 

NUTRITICNfl} STUTTLEL

T??? ntfit.it1(u o‘ ’“Hixfi ?u:v lager: st1llied.lwy ungy imrvesrti-
gators, ard .“ile various plant oococtiors are used 's culture
redia, tire impori¢.133 of 5.7nn7 lodge Cd“Uié exact cccfnneition
of a cu/lturc radium baa aloe been recognized and for phyaiolo_

gical work a rurher of syvt7etic sulrtinns cert
chemicels which are consiiared indispensable f
have been devised. Lolvtions, however, that lwav

for

ner+icnlar set of

1

may not at all mjet tne
contrary,.tle work of Co

1022 (a?) and probably of cth re shows that the
growth arc fruotification vary and that a tedina
best for a particular fun as is not suite? to so

0 taki

.J

ov r, ng into accorn

environrcrtal conditions

éccns (T4), it i“ “‘1:

frportafli.rvdrejr var at
\nrr‘atfiwvs: obtrixwr» ”37"
with a loss Cf i“? color
linked to~3 her in +‘sir
CCf"eifl“irf“ of' " i=‘7l‘l-

a.

t3"'e (30101“ nroihzct

’\

F.1t5-011 t}‘{‘ frllL._H‘-}‘r‘ ;~_ -1”.

1"
wear. ‘3

9‘3

ynttetic

nducln"

.
4

Ho

spcrulaifixui. It

L

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f t

C+

prcncrtiors o

J)

concentratiois for this

I
.gandiifrn“a a?¥?;for E
recuirmnents of unoifier
‘
0 v
ore 1n lTl? (94), lawn”

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'i'YiLS 01.0.14

he salts above .d

a:

(L.
- I

C1"

23
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,.‘ . i \
L CL)1‘.\‘L1IC {-71,

irijds “Ci 5

m

,,~ - u w 4-
print“ :r F I 0:: uh

been developed

lar funcus

\
v

i‘ CY: tit}

1’7 f’li S o

i

l

t

TziltS Cf be?

kncrn: to be

' Hero—

Othfir‘v

‘_ _ o__ -\ x _ 4 ~ _ o o A _'_‘ ‘-
t the different out ogfinite restorse to
f
1.. .U. " -‘ .. i, D .
as 91Cnn by oteVnms are “all (?4 a;
O
re:yw ij’nl frufid }"el:‘Llf: u #1 r 'n1:w* {1*
- b
A r‘ I'- ‘u ' '\ 'o " A. ‘ ‘ x x
in. 4L. l, b-OI (b JLV'N L" ‘.L *3"
ll th~ 'GLJW“‘ of ' lfVW1<if enorulctlrr
fllV: if at, ttueafa t"xw C}21f%p3t p«5 g3uop
ji>r*€v=ancl:. Ir1¢1nJ'V‘ to ;33t v" ir ’
<‘ ‘ (‘F “\ L#1*‘i. “1* s' lids a"3 ow‘atxr' CE“
I
fruit‘/* of fl} fflh"l ur"r invest?-

g

r
\.4..

cht that increasing or decreaoé

 

 

 

 

.3: Haj».

- x; .r...1.H. «316351!

"h
’ \

._/'_,'

obtaired favorable for the study of color product'cz by the

. 4 .- ‘I ‘ \‘c‘ “‘1 ‘ ‘ " ’."‘ -»-« .m‘y‘J. ‘ L's-A
different fungi. The results cl sucr exp>rlns ts ml,nt furt'er

1 r:2t that irdrced t*e ocrrarent var?ations.

kL

Coons' solut‘ou 1* ad, of the follcnir~ inerF lite:
For 100 cc. f medium:
1/? Va1-cs 5 cc.
3/5 avnrna in 1 cc.
3/: rssao4 5 cc.
Y’S YgL64 1 00.
By arranginf L’s nuibgr of 60- of I/C of user of t?: above

nutrients in a series accrrdinq to the rcethod us ed by the
Committee on Salt Acquirements 0f Plants
aticniil.me°~irc?:LhnumaLlAJcr wacn:.Li the study ci‘ruitriticn cf

.‘

Q H's. .’ LI, we ,-1 .‘ ‘
hl”VY3T plerdaa, &Ku.tuulinflL5LZlfuT {LIJL eroruj: uutdl ‘to [tuxe‘up

19? cc. of solutLun, a r urber of cultu1e media were obtained
each varj‘_r:é by incr manta of 1 cc. of 3/5 from the preceding ard

following cu"*1re. The avount of‘ 1:35.04 was coxstznyt in all the
cultures and while m3 tone trd KHQFC4 were varied both above
and below the amouit iven Ln the regular formula, Asparagin was
only increased. A four figure number was given to each solution
in which t*e first number stands for the number of cc. of H/S
Haltose,in 100 CC. of mediun, the second for the number of cc.
of 1/5 K82? O4 , the third for the number of cc. of H/E Asparagin
and the fourth for tLr;= number of cc. of 3/5 {3804. For exanyle,
the number for the regular fornula of voons Zedium is £511-
meaning 5 cc. M/E Laltose, Soc. E/E KH2P04, 1 CC. M/S Aspara-
gin, and 1 cc. M/S Hgédq per 1C0 cc. of medium.

The ir nsr dLer s were mixed in the correct proportions ard
the necesSary anount of water containing the agar in solution

‘

was added to ma<e 100 cc. of medium. lhe radium was tubed, steam-

 

 

 

   
 

LI. . ,I n.
. Iciutfltf‘rluf‘ ‘1 ”OLI‘ . v1

—r:,/‘-—
a -

sterilized on 5 successive days ard tbou slarted. ibe agar was
added in sucb ar swoon as to five s, in each medium.
it must be added here tLat theSe experiments were '5 ended

as preliminary, since it may be easily observed tbat the

d also tie osmo—

-\
{.1
—-

}

reaction of the different media iiffered as ‘

tic ressures. r“his investication 'howevsr, had to be disconti-
P M 2

nued attth: cfld of guaust,1922, and tbs results of this part

of the work are only offered as a oossible startird point for

A.

V

C‘

sore succeeding irvestifator. It was origiuolly platted ft vary
also tbe amount of Cr304, seepino tbe Sifar constant but time
was not available for tbis eype i ant cit er. Fifteen different
redia were prepared as described above and arrenpeu in tre
following triangular form:

Haltose

1st figurel
7511

#4?-
6321 5411
8.41:”!!-
5331 548 5511
#4 as on
341 4451 4521 4611
*% $4 an %%
3351 5441 3531 562 3711
as so M4 we re

Asparagin Potassium Acid
3rd Fifure. Phosphate.
2nd Figure.
Since this experiment was intended to test the possibility
of inducing Variations in color acoordinr to food supply, it
was thouobt best to use solid redia fibers the color could be

better observed and compared. It was toerefore, impossible to

determine a:z:oxmts of frozfirtb, although in one case a set of

 

y’l Ills! I“ \

lg

 

liquid Kedia was prepared in 60 cc, Erlenmeyer Flisks contain—
ing eacb 20 cc. of tte particulir neo‘um, and Colletoi ricum

lind~nutbi‘rum Exp. Sta. Strain II grown on it. iais experinen

1,1,.

will be des scribed in detail further on. Leftoria apii, Colleto—

Y

tricbur linr'ru+binrum Exp. Sta. Str. I & II and Cladosoorium

.L
n.- -—

 

 

—--

 

-0...‘ -

fulvum botb par nt Hid variant were grown each in duplicate
on tbis series of solidinedia. its following is an acooun of
the behe vioar of eacb fungus on this set of media.

83PECDTF APII. Tbere was no notic able variation in color

.5. ht‘

of this funnus rrth on a set of nedia as cescribed abFVO. rb‘
fungus was always black witb an abundarce of pycnidia. Ebere is

difference ir tFe amount of growth in tae various

03

perhaps
Tredia‘trzd tris can be easily ob Jrvtc in tbe picture of the

set taken seventy days after inoculation. (Plate 1’.) ”he
darkest color is towards tLe top of the triangle wbere sugar
was present in the greatest proportion, 5421 and 8411 being

the cultures makins about the best frowth. It may also be seen
tba t a sli~bt white tuft anpeoird on EU 1 and 3621 of tbe base
line of cultures. The 1ecr3afo in tbe proportion of phospborus
seems to have influenced rrowth much more effectively than the
decrease in tbe proportion of nitrogen in tbe form of asparagin
susar and vapn981um beino constzw t. Potassium varied in tbe sane
way as phosphorus since KHSPC4 was the salt used.

COLEBT 1"TCHU" LIWD"“""T“rU” ixp. Sta. Firains I a IT.
grown each on a set of nedia as ;‘ecriboo above did not show
many diffenercoc. The redia were in both cases inoculated with
sroros and t.r e cilttri; were allowed to frow unu r natural

conditions, side by side in the labO‘ator‘. ihe piotures(Plate

V 5': VI) tel-3n seventy five dogs aftfi; inocula-
rive El oood fifty; of tfxe exact,zflzpeezvm cs. inservuli.zrith tfma
characteristic srlmon pink color deVelonfed rorually on all
of tbeu, independ'ntly of tbe blacc color, as forexanfle on the
excess Nitroqen sine tbe blac” celcr developed very uucb later,

(VJ

' . ‘ -' 1': -V " r* “ . - ‘f
the acervuli aooearlns first. lie Gan use tie care w1*b t}:

Lecriar reoium wbicb, as nentioied previously is Coons rediim;
with five times the snourt of nitroren. lbs most rrowtb scoured
on the sofar—PEOsfihorus mid: witb deer atlflf intensity of
color as the arornt of sugar decreased and i”Pics-51>}?orus increased.
Yet a sreat nuxbsr of wbite tufts w: e seeL. :everal wbite
patcbes were observed in culture 5511 (Norrsl Coons fiedium)
of s rain I but on beinv trersfsrred to Cat heal afar, Corn
fieal Aver ard Corn Zeal Flasj they invariably drew black.

[be var'atiors in t*e svount f prowth nty be betfer
studied in tbe liq:.:.id culture-e e>:p<‘z:rizn.,22':t to be described later .21.

CLADOSPORIUN FULVUL was wore resoonsive to such treatment

 

than any of tbo unei under study. Both the brown and the white
form were frown oach on a set of media as described above and
the results were very interostinf; esoecially the variatiors

ir color. Ibis funcus rs“es a slow and rod rately abundart
growth and it see s tbat tbere was enough food for its
developrent in all the c ltures, since there was very little
variatior in t*e amount of trowth. ibe orly variations more

in tit color of the aerial part of the colony, tbe color or
absence of color diffusing throurh the substratum and tbs

in color of tbe subterfed srowth. tevertheless tbe

differenc

\i)

growth on media 5491 ard 3441 seemed to be a little more

abundant than on the others.

 

 

Juan... Ux ‘ T

Referdirv'ifive color difffinshra throUTr ceraairzrmxtum it was
found that tbe color on Cat Jeal Afar ard Corn Heal Afar was
marfoon and a similar piffiont appeared on nodia 4341 (Van Eykea
Re ) 3441 (FainVVan Lyke Red). Both Lhasa LeCiu are at the
Nitrogen corner of the triangle. In ire rest of the redia the
submerged growth was of a brownish OlirVB color tinged more
or les. Hith oohraceoos tawny especially toward tre smear side.
r“he color of tbe aerial part of t’e colony varied consi—
lerablv according to tFe composition of tbe medium. Ir rereral
it we browrish towards tre E’llffitr' column, pinkish towards tlie

‘ '1

nitrogen sioe and olive towards the Potassium—Phosphorus side.

The intermediate cultures merged one into the otter. Table IV
gives the colors observed on the different nedia. The first line
in each czse is the surface color, the second the color of
tbe subrerged srowth, the third variations of surfaCe color
its fourth tre C 10? diffused tirouoh too medium.
all! ij TABLE IV on page as.)
The number after each color ref rs to the plate in Ridgway's

color standards.(d3)

a
|.Jo
3
LL
Q
I..
a

White mycelium appears dos 5331, 5491, 4431,
an the sugar-nitrogen side of tbe triatrle.

"The white strain of Cladosoorium folvufi was also grown 0o
a similar series of media. Table V gives tbe colors observed
on the different media. The growtb was uniform in size of
colony, 1/2 to 1 on, is dirweter, except on 3351 where the
colonies were fron 1—3 mm. in diam. The white character of tre

variation was preserved througrout unchanged, exceot that a

yellowish tinge developei towarCr tbs sugar side, purplish

 

. infill-liralbfiouhufi $35.31 Ionian: u. L, ”V

‘|\s

 

TABLE IV.

938-

Oolor Variation of Clsdosporium fulvum parent strain
grown on Coons Medium varied in composition accord-
ing to the triangular system.

 

I. Odwsucms Tawny

a. 8?“!an 0i3v¢ 30

Sugar.

  
  

 

6321

 

Logmadwcut

 

 

 

6411

 

Lqu Ytiiow 3.0
Lbhwnhk Olive 30
3.9031. Olivc BMW 4°
4- LCqH vam min

 

 

 

p—Afiél

5531

 

..5ouovdo'o oi?“ H.
afivum'dx Oiivc So
$.W‘m'i1 -

 

 

 

 

5421

 

i. hotel‘s Coiov 30
;. fivowmisk Mia 30
A,“ kit.

 

 

 

5511

 

v. ng7 ‘fdlow 3o

1 . fivowm'sk OVA 39

 

 

 

d

l-anifln olive .0
s— 9“? Wu (m1
‘LYMn 91“ K“ ‘3

I_?.‘| evoT'sb Vin“!!!-

'-%uk 1‘3an *0

1.0.1 Olin. +0
3.N\n§\'\

stqidn olivs t“
PWKK s 39

6+ DIM. 30
rsVvau'Sh ‘Mow 30

 

 

 

 

1m

 

H74V0nzi0 {u 11
.Mnish (mu 3.

 

 

ifiggin

 

 

 

3441

 

 

I. f.\. “disk fink:

). GVMnisH ORR

3 . . . .. ... .
J- (“.3 ‘03)}th

 

 

 

 

3531

 

LGn-‘(sin 9kg 4'.

1. flvomsiain ,, bo

 

 

 

 

 

3621

3711

 

 

- I-yuk OiI'H Bus “i
bfivwnist QM.

 

 

 

u' {U Bvowm'sk 0h»!
avowm'd‘ Dim. Jo

 

Phosphate

1. Surface color.

2. Color of the submerged growth.
I. Variation on the surface.

4. Pigment diffusing through the medium3

    

 

...! - Ekals Li. ...-I. .:.,ul... . L 1. 1r
, , H.344.“ .ul..W., . 1, (Luniyg‘u.
uYEQ-hi‘a: [Iris—ul‘ . l

.33.; 1 NT:

Libikmbhnbrhil

   

. . . . - .,
. Ll: g . .:.»! n5 11...- .a.

 

1??

 

TABLE V.

-39-

Color Variation of the Variant of Cladosporium
fulvum grown on Coons Medium varying in composition
according to the triangular system.

Maltose
7311

 

9.01.0!“ Co‘ov N.
p.5uuki‘mu 6mm”
Pi

{fivowm'sk O‘Hc 3°

 

 

 

6321

|.BV?Y1HM *0
kaa.
P.FewnCoiov Jo

 

e411

 

Uflkv‘ Bu“. 4 o

‘I i . - . .

3 , . . . .
JDVNJW mm 6“.“ 4

 

 

 

 

 

 

5331
I. “fit

;.Couee “wk M.

 

3,mq‘\1iv\ouoms 2‘

 

 

 

5421

5511

 

 

LSced‘c“ fink '4
1. W11
3, WOVM Stfs'u”

 

 

I. WW“
g. P.“ Oh“ 6" +9

)- Bvovmidn 0‘“; 30

 

 

 

 

 

 

  

 

 

L LMovs‘N‘W 3 A -------
g 4341 4431 _J 4521 4611
.. wmt .. Wm. .. Wm. .. wkm
_Q.\fi \i\o¢ 31 4 ...... ,. . . . . .. ...

   

3. (new. “0‘“ a? sflchqu-heu

*.0\Lv1 R1311 1......

 

 

 

 

LCEnnm-um hm".

 

 

 

 

3. New amok“ 30
‘1- BVMmh‘xO‘k‘“

 

 

 

 

 

 

 

 

3351 3441

R“ 1. WM\‘\\

. .. .. 1. eo\\\;\0c 3')
1“€no¢an\5J 3.1?nouws'vh‘k 53
“oh". {3 *.--.. _...
ragin

3531

 

“whit
.. ?.\._\s\.«. ’1
3. Mm (Mk1 5‘

 

 

1. Surface color
I. Tings

3621

 

 

 

I. WW“
., 9.1. sun (m u.

3. CONnRSW (”wk 5

3711

 

 

LWHH
2 .CH ewe “luv '8

LM. 01M. 39

J. n '1 30

 

 

Phosphate.

3. Coir of colony seen through the slant.
4. Color of submerged growth.

 

 

 

ill! a. lull» r' l: 1‘ x1
,3! a

1;, i \

..LtIVkI‘II, . . >1. -
. . \itl II¢¥LZ . 5.1,

. ... I il lx.I«A.lA.-t
n» . 4.... 1.1.: H I . ..IVI. .
«(5.3. r .1! . v. .1...” v t .. ...... Q . ....
- I. ‘ «1.4. 3. .. . . . ,
t A. 1.. x 4... .. .
, . I ‘

 

towards the nitrc“en side and yellow—pink towards the Potassium—

phcsphorus side. Qt account of tTe purple tinge the w*it3 was

\L

more striking and cle.' font the niLrOfen 5r

~~“° Table V on P&”w 39'

 

LIQUID IEEIA. Only Colletctrichum;lirderutbianum Strain II

 

was grown on liquid media. lwerty cc. of each medium in the set

was placed in a 60 cc. ercrreyer flask and inoculated with

f

.1 cc. of“ a spore suspension of tie fainfus. Ire cultures inz“1.")
duplicate were grown at room temperature on clincstatAfor 76
days. At the end of this period careful notes were taVen, the
flasks were photographed (Plate VID and their contents filtered
on veirhed filter paper. lhe mycclial mat and the filter paper
were dried to a constant tenparcture and reweighed. lable VI

recordt the weights of the duplicates in each medium, their

1 0

average ard dgbulipthhS of LL31? growth and color.

liable VI 0
firms of frowth of Colletotricrum linderuthlanum grow:
on liquid Coons sodium varied according to the trian—
gular system toast? r sit“ a macroscopic description
of the cultures.

Medium . let Ser. 2nd Ser. Average . Description
R138. Riga. ”.1830

_‘-———‘—{_-————‘_—qu-du——n-qb—-—~‘_---——1h--—-—————‘——-—--———on...—-——_—

7311 p 17030 0 23000 4 20010 P ALUNQRWL FTCWth, SHbmorflPd
mycelium dirty white, a
rir* of Natal brown 53‘
irg to ttc ;lass on the
surface of tie medium.

——--—~-— b—----—--Jh_—-----—dy———-—-—-— -’-’———-‘-—--—---_-__-_—---

W"
J —

\J

6321 9.9 4 9.0 l 0.4 . tide blackish brown ring

i around the edge, with
black small sclerotia like
formeifixris scatterwfil‘t” oush
it. pub” rfed mycelium
white.

——---a-‘—JL- ————— -——JL--———-——Ji—— ——————— ii —————————————————————————————

W

 

 

 

 

 

_ .__._~.—..—..._..___.
_—_4—~——__~————-—1~-—‘_~~_~——-

 

.-V-.._.-.nm—“_—.——o—._~—_——.——q—_—_’.~._~—__~_

--——.—_'___._.--._..h-—r—.~_————~———H—— h. __.______..__._,, .-....7...._-——_———--—

 

.
AA __ _ V- ,. _, V... 14- 71.1.. .._7~7 .. __. _. H '—
c o 1 I. . O .
C
‘
, l“, - ___-__ _.—_.,._-_._-.——...._._ -..—...--_,‘_-.._i-—
__———-—7
-—- ...- 41‘

 

Table VI contd.

n41-

 

Iedlum

p

lat Ser.
mgs.

r 7

2nd Ser-
mga.

Average .
Inga 0

Description.

 

15.5

. 25.3

20.2

No ring, no sclerotia,
white mycelium, Boyal
brown patches on the
surface.

 

5331

13.5

Abundant white myceliumo
Concentrically arranged
sclerotia on the
surface. Narrow, black
ring discontinuous.

 

5421

4.7

Mycelium brownish.
Concentrically
arranged sclerotia on
surface. Discontimuous
black ring.

 

5511

10.1

Mycelium pale brown.
Ring indefinite.

 

4341

Mycelium pure white.
discontinuous, loose
black ring.

 

4431

Mycelium nearly white-
Wide, compact, black-
ish brown, discontin.
ring.

 

4521

L. 4.2

5.0

Mycel. dirty white.
Black, compact,1 cm-
in diam. patches on
the surface.

 

4611

 

 

 

4.5

 

Mycel. brownish. Inde-
'finite ring brown in
color with sclerotia.

 

 

.qH-_—-—~4.—<~—a—4—.—-——.——--.-_—-o‘—O~<_———qv—d—--—¢-—o—¢-4—--‘**<—C'—‘——Q"—4—‘—-~4—<—*¢-~—-“——0-l-—O———-—-¢-—u«_‘———.

o o . o .. ‘
O ' 0'- I
—_~‘ __‘..‘_-a-—-———_—~————-.—---~—--—--~——-—-oc—-—. .--—~-- ---..-_-- _.—._ ...--
H
a .. ' ,\ ‘- r - .
n A f O O 0 0 ~. _ g
.
s - .
‘ ~
0
~- ---._H.. -.. , ‘.—--. .....- -- - 4-.., ..- Gn-‘uu.’ —.——-‘ —.—----.—._7--4—7.- -_ .--
3 . .- V
O O C I I C O
O
.
. . .
w _ .
c . u
-. ‘-
0 .
—-‘--— —-—--—.—.-_..-—-~‘— n-o-——--o-—--- w-ug- .---.. --.-- -—-——-—“—— —>-»«---
I ‘ ‘ b. .4.
. O . .... . O .
a
.
.
n . "f
O
.
.I .
. .
,-. . . ..-—......- .._‘— , 0-- -—-<' ~---—-—-----—--~—.-—c-¢—‘- ~-—- -- -..- .——.—-.--_ .-._ o——-__-
‘ ‘ - f ...
' 1
o 7 - - O O 0 0 .I . c -.
h 0 O I
. .
A
D
\
-.. -.-—-_--—-~--——- .. ~..———-——~- —.~.---——-~ —-——-¢--- .---- -— “-4.—-c-. -—-—-—-. ..-
g . A.” -.
l
. . I O I. 0 Q o ,_
.
‘ 2
O
C .
_____-_._._----- ... ---- ‘--"" --" ’-""”“ "" ' "’~ ' D ~~ 0—.--- .:--
_ . , ‘.
Q I O O C O C
, I
I' ‘ I l '
o. a . Q
I . i
' 0
. ' a .
o\
v
. .
_ . -.- --.~--__-‘-"-_-‘----‘-“~-_‘*~~-‘~—fi‘M-fi-‘ _
-- ---—..__. 7... _._..—_.‘.n -
a - ' '
. ‘ \ ' r O ‘
. . . . . . . _
. , ‘
O ‘ .
.
O
.
v’ t ‘
C .
—- .-__._‘.._._..‘----._.- --—¢--—--‘ .--..-.-. _—-.-.---__-
--——o------—-v--—_ '-__.-,.-'—

q wW‘ov'nr-v“
v

 

‘. —; —-———_~---————-—-- -——- “.-—-"’“ "’".'-.'—'--*—c— .—. -~-- --—---_ -—
.. -----cc—-—I ---~ -

Table VI contd.
-------- r----------w----------rr---------v--------------------a

Medium , lat. Ser.¢. 2nd Ser. l Average , Description-
- ------- q ------------ p- ---------- Jl- --------- 4 ———————— c- ——————————————
3351 2.0 p 11.0 t 6.5 V White mycel. Thin
ring with sclerotiao
II- ------- run-- ------- 1P ----------- iL --------- ‘r --------------------- n
3441 s 6.4 . 2.1 p 4.2 . Mycelium neatly white.
Thin ring with sole.
J rotia.
--- ..... up ———————————————————————— p ———————— c i —————————————————————
3531 l 2.0 o 4.8 r 3.3 . Mycelium brown. Thick
i brownish ring.
I
3621 9 3.0 0 4.0 , 3.5 . MyceL. brown, ring

indefinite, sclerotia.

- ------- q. aaaaaaaa --‘r------- ..... r. ......... u. ------------------- --—

3711 0 coo 0 209 b 209 a Mycel. brown, ring
J J indefinite, sclerotia-

h ---------- L ......................

 

 

 

 

The mycelium from the different flasks was examined micro-
scopically. The black or brown pigment was found in the mycel-
ium. The bodies referred to in the table above as black sclero-
tia were tiny tangles of mycelium. Sporulation was found corre—
lated with the color. Brown or white mycelium showed no spores-
Transfers from several flasks showing white mycelium on Cat
Meal Agar grew invariably black. This seems to indicate that
the variations in color were not fixed but were due to nutri-
tional factors, whdhh prohibited the formation of spores espe-
cially towards the Potassium-phosphorus side of the triangle-
Sporulation seems to be linked with the color character. Lack
Whenever the aeriil black ring or the black sclerotia were

formed the mycelium was white and spores formed, but when no

 

 

 

_ . . p
1
.
_
n a . no
.
.
l
.

_

.

_

_

_ _ .

, _ _
_ l _ _ _ i .
_ 1 .
. .
i In: L. [r I. u. :1 4......» NE? .ISTlitiiIIIwTIL Lula-kn! 15..., i L. P t
D‘ . x i n Ill Jr \ . I l 1. yr .
. . . ..- H . r . i: . . } . . .. L. rL._.,.WLan . .....mer.
.L' \ AI. ” I. ,u v
I - I ll! ll! I
l 4 l I .7. . . .u: 1w. . I... .....I ll'l
. 1 1 us». 1. .1} .
..\u a r. 1,: V

 
 

 

-43-

DIAGRAHS illustrating the regions of greatest growth and
development of pigment in cultures of Colleto-
trichum lindemuthianum gronn on Coons e um
varying in composition according to the trangu-
lar system previously described. .

Maltese
1st figure.

mm Host Growth.
Black Color.

 
  

 

4.5
*4!”
.- . . - 4611 .

6.5 4.2 "‘“’ 3.3 3.5 2.9
9*” 5*4’ “*9”? «IQ-”l. *4!“
3351 3441 3531 3621 3711
Asparagin

3rd Figure- ; Potassium

Acid Phosphate-
2nd Figure.
WRS
*4”?!
7311
WRS HEP
“-91-!!! 5%!"
6321 6411
NS? 558 BS
«‘01»!!! *fi-fi‘l‘ INN-*4!-
5331 5421 5511
WDP IDP WP BS
{MR-*4!» *9”!!!- fififlfl- fififlfi
4341 4431 4521 - 4611
WRS IRS BRP BRS BRS
3*?!” sa-r-ey- su-ms «I‘ll-3* 4944*
3351 3441 3531 3621 3711

Legend: W: White mycelium
B: Brown mycelium
R: Ring present
D: Ring discontinuous
S= Sclerotia
P= Patches-cf brown mycel. on surface-

 

. .44.

such structures were produced the mycelium was brownish and
nod spores were found. Lack of proper nutrients more than any
other factor caused these variations and sugar and nitrogen
seem to be essential for both color and sporulation.

licroorganisms in general are negatively phototropic under
strong light. Direct sunlight is detrimental to most bacteria
although some fungi are resistant to it. Fungi grow well and
fruit regularly in diffused day light, which in many instances
seems to be an essential-factor for reproduction. Coons (24)
found that with Plenodomus fuscomaculans light is a factor 11
directly concerned with pycnidium production, the fungus refus-
ing to fruit when kept in the dark,irrespective of nutrition,
aeration, substratum or strain. Makemson (54) found that light
had a retarding effect upon the growing germ tubes of Clado-
gporium fulvum but the ultimate length was the same. vegetative
growth and sporulation was also more profuse when the fungus
was grown in the dark. Stevens (83) observed slightly less
zonation and less aerial mycelium in the dark than in the light,
working with Belminthosporium sp. grown on Petri dishes. Smith
'ohd Swingle (81) report that diffused daiZlight affected consid-
erably the color of fusaria and Sherbakoff (78) found that
diffused daylight intensifies the color of Eusaria, while intense
light dulls it. He did not find light influencing other chara-
cters in fusaria. Older literature reviwwdd by Coons (24) and
Harshberger (41) is Just as contradictory. It seems that differ-
ent fungi vary in their response to light of varying intensities
some vegetating best in darkeness and requiring the light
stimulus to induce fruiting, others growing and fruiting best

‘ ---.- — ‘3 - - - -

.ilill'ihunaiflhuug
.. u: . .43 . .

 

-45-
in diffused daylight or in darkness.

In order to determine whether light had any effect on color
changes and variations of the fldngi under study, four sets of
media as described under Nutritional Studies (p. 32) were
inoculated with the white and brown strains of glgdcsporium
fulvum. A set of each strain was grown under a bell Jar
completely darkened to exclude light. A second set of test-
tubes arranged and fastened on the walls of a battery Jar,
covered by a bell Jar , was exposed to strong diffused light
and gradually rotated to overcome differences in intensity.
Air was circulated through both bell (are. The Jars were not
disturbed until the end of the experiment when the colors of
the cultures in both sets on each medium were studied and
pictures taken.(Plates 8-11.)In general light did not seem to
szect the color or growth of the colony or the number of
variations. A detailed description and comparison of the
cultures on each medium in light and dark is given in tables

VII, VIII, and 11:.

Tables VII and VIII are diagrammatic, being further cnmple-
mented by table IX and Plates VIII— XI. Slight variations in

color as given inthese tables were neither consistent nor in
a definite direction,to be taken into consideration in conclud-

ing on the effect of the factor under study.

 

 

LL n Hildgihmggfl ."L I“.‘

 

 

    
    
     

TABLE VII .
Color Variation of Cladosporium fulvum, parent form,
grown in light and dark on Coons medium varying in
composition according to the triangular system.

MALTOSE.

Light 7511 Dark

 

Lockvauous‘swnY l- (5%“ o‘u‘vt 3°
1-Bvowv‘lsk olive. 50 1. Bvowm‘s“ OhK 30

3. W whiit wiovfizs. 3_ 3 W\m'\e M‘-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

  

 

 

 

Light 6321 Dark Light 5411 Dark
\.Gvay§s\'\ OHM. WLGvawisM O\u‘va Ma LOLlwo‘LeomsTownyISGrko-Yc'gk 0‘5“ 1%
3.. . .. .. ‘ ‘ _ _‘ Lavowm's‘a 0““ 30 BVDWVU'sln 0" 50
3.W\'\HL ,,,,,, 3. i WAX Co‘om/ Wkih l 50‘.
‘1 ............. q, d .........
Light 5331 Dark Light 5421 Dark Light 5511 Dark
«Venn 0.09. By. m ‘MN‘UYK' Byawn 13 I. Bgskgr 1“ LSchovda'o (funky/1.1 1.?vast 93v. 3 Ll’fivussds fivewn 3
1.3.qu OHM stex’ 51 LEW“ 0““ (3me 5| L ________ 2. _ ....... 1 avowNSV Ol'"« 50 2- mewvu'sh GIN-L50
I
a. -..-—.- 3 1W4“? Lelia-W; sill-1W» 3- MM“-
* ------ “ 1+.------' 4...-..” LU...“ JJ.BYDWY\. Ofin‘L-‘HH‘
Light 4341 Dark Light 4431 Dark Light ‘ 4521 Dark Light 4611 Dark
' MWMW“! BM" '5‘ LMUW‘M‘1 Bvowv‘ ‘5 ;,mumm\1P§/V- ‘5 \‘ memn‘ [3"- ‘r v I. bfidav '14 ‘- 56‘0"“ 9’41 / W /g/\/J
1--........1-.. ....... v ;.—-~-~--- z. ------ . g..---~"-- ‘ 7M """" \C’F/ V“
0 0
5 W‘mW'L. 3.N\m‘h, S’wim'h, 3‘ WM, S‘inflgk 3. WWW, 6V / 6V /
.1, Covey PM.“ w} WCmoi, (sww MW“ PM. ‘4 J _______ .. 4” _ _ _ w. _ _
Dark Light 3441 Dark Light 5551 Dark Light 3621 Dark Light 3711 Dark
34, 'EQV‘ BV'V;“°L;‘;V‘ Vup‘hnouoms (W055 O‘I‘K fivovm 4.0 16‘5“” M’ L stkcv 3“ 1. Raw UmEev 3 I. Raw wau 3 }. Mede‘ nwvvfii l. M:o\o‘ BYOVWOL
4019b“ Pav- do 1‘__._~___ ‘+ 1 ______ 13.”. ¢,.--... a, ...... 2.-...1...---.>---"'
" ------- 9,...-.-- a.vxx1Vu 5,--~-'-- awn/Wis, 3.\V‘43{"¢ 3' Wlfi'flt 3--‘-- J'---"
“_._”. “““ Nev. Nani 3% u}. give»! “Y. "i . HOWKMI‘MMRQ 1‘) J-QOVk Mineramvj J. £\'v0V a“ ‘4' 1’} ~ . - - ‘ ‘ J' -------- 4' — — v '

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PHOSPHATE.
1. Surface coloro

2. Color of the submerged growth

3. Variation on the surface.

4. Pigment diffusing through the
medium.

1,11

 

 

  

  

TABLE VIII.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

  

   

 

 

 

 

 

 

 

 

 

 

  
 
  

  
 

 

 

 

 

 

 

 

      
 
 
   

    

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1. Surface color
2. Tings

3. Color of colony seen throu h the i

4. Color of submerged growth.slant.

 

 

 

 

 

 

 

 

 

 

 

 

 

-47...
Color Variation of Cladosporium fulvum, variant form,
grown in light and dark on Coons Medifim varying in
00mPOSitiDn according to the triangular system.
MALTOSE
Light 7311 Dtrk
1
4. Guam Co‘av )L Naples \M‘ow W ‘
,I.....,_,. ;, Wklh.
aAYLva 00L“. )5 3_ jeuow OEAH l5
4 96/145“! 0‘-GV”VWH 4. QV-skj 0‘?“ (w. V“
Light 33 21 Dark L1 ght 6411 Dark
V1
Leah Ochaasw‘s offah Oohvouous ”7 I. C-‘v‘v‘mwwn 91w“ LOII'H Sufi ”0
1..... O LU..._ MD” 1.---- I:1.WL\1‘"L
3‘ OtLvouous Ovohgtzod‘wuow Riv“ ‘5 5.00invouvms GM“ 3.C.',mo.mw (3%le
d. - ..... 40mm 0‘ GvuMH A'QM‘“! 0L vaix‘fljilfimskYOIfiveew 4|
Light 5531 Dark Light 5421 Dhrk Light 5511 Dark
..WML ..whcit .. Mach finish Pom“ 1% 1 WW. ,, WILM'HT
LCGwlo Fiwk 9—(4 few-u; fiV‘K 3‘1 meg“ finkisL‘ Gnn’fi \Vk\‘\'L 1.. """ ' 1'1" H4“ 614$ 40
10ch Red ll 3.0%“ R193 17 5,\f;wouom'|owm7l7s 00in”. RVJ‘ 17 sflsohdheoiov 50 3. isshalim (301m 50
.\ .Qmsk\{o\.GV21V1*\dn Qvékj 01.0w m Jrfit‘ou‘a. ;‘1 OOVKOHVL 1+0 4, DokalM. 1+0 4'. Dex/K Ohm. ‘4 D \-
Light 4341 Dark Light 4431 Dark Light 1 4521 Dark Light 4611 Dark
l th'h. l. WMH, - 1. Wim'h, I. V\"L\\'"L )- Wkii L. ). NHL» !. WMH’g JUW
‘ . - ._M ...U.. K/
2- (\‘\0L. 15 1. ()oh \\'\0e 37 2_T\\\¢u\ (5%“ 4° 1.6942 Moo 37 1, . . i 1. . q \[0 /
5.99vkflo‘loow “~1ka 3, chdvok (in) 3% 3.\ovK (imouowflrs 3«O¢¢FCOV;V\*{‘-Rbd17 _3_ FeVYV‘q‘,WO‘/“§ ‘J 3.€vv\$sv'a\n RV; 9‘7 1‘1“?“ SUlO?V 3 g
Av, ,Vk\y.o\§a.v. Ruiz] J.E2qh\gw\ Elf-1191 J‘Hofo PM. sq J.W5£\'aw a” \‘3 J».r25V°WV\\ISL\ (MM-£30 4, ®°VK Ohm 4° 1. vowvx. . 30
D rk
3351 Dark Light 3441 Dark Light 5551 Dark Light 3621 Dark Light 3711 a.
‘ - 1’ y .‘ V. 1‘ I,[-' t u
"€°\‘\{iwouum\{\od Levis (i‘wOuomsmocdu Licw‘a Evowu 59‘ t. WMH, \. Wth I. Wha‘fi " Wk“; x‘f’qm 8"“ 5’; ”C (“if (5 H 5 qh B it If
L . M ' ' ‘ “ ~-—- 7. ... .. 3. ()ok \§\ot 37 1.Q1qNBv.l/fwoua§é 4. «090103;. FMX 1L. }- C's” ‘“°3}L:‘C:V\JUi-Odwatw\u7oww7 i. o-‘x‘ — ~ r‘ 2 ......
Alfiovkww‘IUW‘KA‘tngSQoVKY‘vwoLuusgw-(i 5.MJAV$ PDVawM l§fl>vm AAASK‘/6M?]IL1(7 5. QCVW “WW" 15’ 1me PM" ‘3 ‘ 3' PVMSSCOV‘ «VA 3‘ L‘Vev Bvawy‘ . 3. ‘0 Vifoajvky 4 -----
afgevkov't "ro 4'Q0VKOhm 40“ A..._ 14......“ v1 4W ‘1" “““ ”M "L V0“ 15 '
PHOSPHATE.

 

\ . \l. .
all! 3.. flfluJéi-Iq -

.r"

a
i -

 

c.48-
TABLE Ix.

Comparison of two sets of cultures of Cladosporium fulvum
Parent and Variant grown on Coons Medium varying in
composition according to the triangular system, grown

in light and dark.

Compo-

sition Description.

of

medium. Parent Strain-

7311. L.(Light) and D. (Dark) are very much alike. Good
growth. The color is the same, except that the
buffy olive found in the center of a few colonies
in L. colors most colonies in D. especially towards
the base of the slantoOchraceous tawny is seen at
the margins of the buffy olive colonies of D.

6321 Remarkably uniform. No submerged growth.

6411 Quite different. D. is composed of a large colony
with white aerial center, uniform in color. Abundant
spores of same color. L. is composed of small colonies
covering the entire slant, tawny Olive in color with
a few buffy olive and one white.

5331 Remarkably alike. Reddish brown and Van dyte Brown.

6421 Alike. L. more brown than olive green. Both show white
variationso -

6511 Exactly alike. L. has more white variations thanD. No
discoloration of the substratum.

4541 Alike. One large spreading colony in both.

4431 Similar in surface growth and amount of white variations-
L. is made of one colony and shows color in substratum,
D. is composed of small colonies and shows no color
in substratum. At dry top of slant both show buffy olive
growth- -

4521 Almost alikeo D. is a little more brown and shows more
white variation which is not compact but loose fuzzy
growth at the center of the colony. Submerged growth
and Ink pigment through medium are of the same brown
red color.

4611 Broken.

3361 ‘Cobor alike in both. Colonies smaller in L.

5441 About the same. Vinaceous in L. rather brown in D. Color

through medium more pronounced in L,

 

 

 

 

_-49-

TABLE I! Contimuedo

3531

3621

3711

7311

6321

6411

5331

5421

5511

4341

4431

4521

4611

3331

3441

3631

3621
3711

Surface color and type of growth similar. D. shows more
pure white variation, a slight white fuzz on one colony
in L. Color through substratum the same in both-

Surface color the same in both. Colonies in D. smaller.
10 substratum color in D.

Clive green- Mostly dried.

Variant Strain.

D. and L. are almost alike in general appearance- Colonies
large ( 1 cm. in diam.) fluffy, raised. D. is slightly
darker and has a surface colony of pure white. The under
side of the colony as seen through the agar is yellow
ochre while the growth into the substratum and in the
water of condensation (submerged) is olive green.

D. and L. exactly alike. Mat like single colonies, white
tinged with pink and ochre. Large guttation drops in

the middle. No submerged growth in L.

Uniform in shape of colony. L. is strongly tinged with

ochre,more intense at some points than at others. The
edges of D. are rather olive buff with the center white.

Identicalo

Shape and form of colonies alike. Both show the buff pink-
ish tinge, L. being lighter, however-

Small rounded colonies, white tinged with green. 5one
pink in De

Shape of colony the same. Colonies covered with small
drops. Tinged with violet, more so in L.

White colonies slightly tinged with purple. Under side
of colonies of a mixture of purple, brown and olive-

Fluflfy colonies, with no drops on the surface. White
slightly tinged with pink and ochre. Under side of
colonies rather red in D., yellowish in L-

White growth in both.

‘11]!0 e

D. white, L. brownish purpleo

White colonies, purplish tinge.

Alike. Tings slightly varying.
A1 1k0 e

EFFECT OF REACTION ON VARIABILITY AND COLOR.

The reaction of the substratum has recently been reco-
gnized as one of the most important environmental factors
influencing the physiology of plants. It had been formerly ill
thought that adjusting the reaction of the medium used,by
some sort of titration,to a point above or below the turning
point of an indicator, usually phenolphthalein, was a satis-
factory method of securing an optimum reaction for’microorga-
nisms. However, with the discovery and perfection of methods
for measuring the active acidity in media, investigations were
conducted which proved that the Hydrogen ion concentration
should be classed with temperature and,moisture as an important
environmental factor. It was also found that besides influenc-
ing growth in general, the reaction has an effect on color pro-
duction, fruiting, zonation etc.

Sherbakoff (78) in 1915 found that acidity induces the pro-
duction of red color in those fusaria which make a grey-white
growth. on neutral media. Acidity also lowers the rate of
growth and makes zonation prominent. Fungi are able to with-
stand a comparatively wide range of pH variation. Meacham (56)
in 1918 found that Lenzite sgpiaria, Fomes roseus, Merulius
lachgzmans and ggnigphora_cerebe11a would grow from pH 5:1.7
on the acid range. Webb (91) in 1919 studying the germination
of spores of different fungi at different pH found that the
range of spore germination with respect to the reaction of the
medium is between pH 2-10 with maxima at pH 3-4 and 7. Johnson

(46) in 1923 found that the reaction best suited for the
growth of molds lies towards the acid range of the neutral

 

 

- ‘ -51-
point. Goes (40) in 1924 found that Fusarium eumartii is
capable of growing under a wide variation in the H-ion
concentration on 2% dextrose potato agar. At optimum tempera-
ture no appreciable difference was seen in the growth on media
varying in pH from 4.5 to 8.5. Hopkins in 1922 (44) found
that by adding 3 drops of 50% lactic acid to 20 cc. agar,

a strain of Colletotrichum lindemuthiangg_which produced few
spores in neutral potato agar, sporulated freely.

In order to determine the effect of variation in the pl
of the medium on which the fungi under investigation were
grown, the following experiments were undertaken. Since the
variations observed were in two directions, via: a lack or
modification of pigment and the suppression of sporulation,
it was thought best to use two kinds of media; one favoring
fruiting and the other favoring vegetative growth. Coons
medium (24) was found by its originator to actually favor
fruiting and the behaviour of Sphaercpgis maiéprum in the
present investigation also indicated the tendency of thisim'
medium to induce spore formation. Richard's medium E (67)
has been frequently used by many investigators as a general
synthetic medium with good results. The modification by
Karrer and Webb (49) was used in order to avoid the precipit-
ation of the phosphate in the alkaline mlmbers of the series.
Liquid media were used in this experiment and the filter paper
cone preparation-dish.method was adopted as reported by Coons
(24) and recently by Bonar (12). This method consists in
growing the fungus on a filter paper cone placed in a small deep
petri dish (preparation dish) with 10 cc. of medium. Schleicher
a Schull No 595 7 cm. in diam- paper was used. The deep petri

t ., 4333.59? . .. ,w
I, . . . . ,
y T Li. Lu... Ln :5... .{lrF .1... ..égzgurx , V, . . ., - , w
w w. I I v . . .‘lm,rv ..i \uv: . van-(ll
, r w .

 

 

\ :‘y y.(1(, u .I u L ‘5.L,I.:u\y»y.l.v,.v ,-
- . .. with. If V .. . .., , .\
> , .... .,. Kari. I , ,u. . . h Ira...
{I‘DI's I . .. .n . ... . .. ...: .I I4 .
. \lti... H. 1
...;‘ln .‘ . .. , . ,..h.x.n..!..lyyiun»ltt I

 

-51h-
dishes about 5 cm. in diam. and S cm. deep, were again pro-
tected by placing them in large moist chambers or under bell-
Jars. Under such conditions the fungus grows on a solid sub-
stratum of pure cellulose kept at constant saturation. Both
the white and the parent strains of Eladosporium fulvum and
Colletotrichum lindemuthianum Exp. Sta. Str. II were grown
in duplicate series of 10 different H-ion concentrations varying
from pH 2 to 8.4. Besides the paper cone cultures, the fungi
were grown in an exactly similar series of media in test tubes
with the proper indicator added to the medium. The purpose of
this experiment was to determine as far as possible the shift-
ing of the pH by the growth of the fungus.

Previous to starting the experiment the titration curves
of both media to be used were determined. Karrer and webb (49)
report a titration curve for Richardfs medium E modified, but
the present writer was unable to duplicate their findings.
The pH of Richard's solution is given by them as 4.6 while
with the particular chemicals, apparatus, etc. with which the
writer worked,it was found to be 5.0 . This finding necessié
tated the retitration of the medium. Table.x.gives the number
of 00., the normality of acid and alkali, and the number of
00- of water to be added to 25 cc. of double strength.medium
to make 50 cc. of medium of a desired pH and of the proper
concentration in nutrients. It was found that by using weaker
solutions of acid and alkali than those recommended by Karrer
and Webb (49), a smoother curve could be obtained. Fig. 1
gives the titration curves and Table XI the titration values.

The preparation dishes with the filter paper cones in place

 

, . l.'

‘ 'A .I 5.. A 1*.E: fl.‘ A

...i.‘
,. I5 . 4 .
rvuzr ,L .. 3.... .4 1.

1.
w) . .. if

 

 

TABLE X.

Co. of Acid or Alkali and Water to be added to 25 cc.
of double strength medium to obtain a series of media
of varying pH and proper concentration of nutrients.

 

 

 

 

 

 

 

 

 

 

 

pl'L cc. HCl l Normality} cc. NaOH 1 Normality. cc. H20 . Volume
RICHARDS ‘ __
2.0 11 l/BO .. ... 14 50
2.3 7 n/eo .. .. 18 so
3.7 1.5 " ** ** 23.5 50
4.8 .25 "' ** ** 24.75 50
5.0 ** ** ** ** 25 50
5.8 ** ** .5 3/20 24.5 50
6.4 ** ** 1.0 " 24' 50
7.0 ** ** 2.5 "' 22;5 50
7.6 ** ** 4.0 '" 21 50
8.4 *s ** 6.0 '” 19 50
COOKS . w
2.4 20 l/eo .. .. 5 50
2.8 8 "' ** ** 17 50
3.2 4 '” ** ** 21' 50
3.8 1.5 " ** ** 23.5 50
4.2 .5 " ** ** 24.5 50
5.0 ** ** ** ** 25 50
5.5 .. .. .5 ll/zo .2445 so
6.4 ** ** 2.5 " 22.5 50
7.0 ** ** 5.5 "' 19.5’ 50
7.6 ** ** 7.5 '" 17.5 50
8.4 ** ** 9.5 '” 15.5 50

 

 

 

 

 

 

 

roof.»

.6 V

.3.

.12; ... $5....st 0.. afi‘E. 3|

In. .1

r [V
i Ail A:
a, v.. »\ CI‘ tut]; ,
i r .
_. -1 n .75. u

 

 

TABLE X.

Co. of Acid or Alkali and Water to be added to 25 cc.
of double strength medium.to obtain a series of media
of varying pH and proper concentration of nutrients.

 

 

 

 

 

 

 

 

 

pl? L cc. HCl 5 Normality. cc. NaOH . Normality, cc. H20 . Volume
RICHARDS T _ ' __
2.0 11 ll/so .. ... 14 50
2.3 7 n/eo .. .. 18 50
5.7 1.5 " ** ** 23.5 50
4.8 .25 ”' ** ** 24.75 50
5.0 ** ** ** ** 25 50
5.5 .. .. .5 3/20 24.5 50
6.4 ** ** 1.0 " 24' 50
7.0 ** ** 2.5 "" 22,5 50
7.6 ** ** 4.0 "" 21 50
8.4 ** ** 6.0 '” 19 5O
coons . . . __
2.4 20 n/eo ** .. 5 50
2.8 8 " ** ** 17 50
3.2 4 "” ** ** 21' 50
3.8 1.5 '” ** ** 23.5 50
4.2 .5 " ** ** 24.5 50
5.0 ** ** ** ** 25 50
5.6 ** ** .5 5/20 24.5 50
6.4 ** ** 2.5 '” 22.5 50
7.0 ** ** 5.5 '” 19.5‘ 50
7.6 ** ** 7.5 '" 17.5 50
8.4 a* ** 9.5 ”" 15.5 50

 

 

 

 

 

 

 

 

. . . u n a . n . . u n u .
t
. u
c
v
. . u o . .
.
e
. n . e a a . . n
I» .
IL
I, ..tnlulabivi rt: . N. i... “21.:11‘2 u \.. n .
n‘ll ’I‘A‘. V v. .. .(v i Z .aY r
_i or. A. I ‘1‘? .3111!!! 1 a y .ylb.v I- v
I

 

054*

TABLE XI.

TITRATION DATA FOR COONS AND RICHARDS SOLUTIONS. cc. of (
Acid or Alkali and Water to be added to 25 cc. of Double ‘
strength medium to give 50 cc. of Medium of a certain pH.

 

 

 

 

 

pH } c.c . cc.Acid.oc.Alle pH l cc Hgotcc.Acid cc.A1k .
320 1/50501 N/20Na6! x/50301 N/ZONaOH.
C 0 0 I 8. R I 3 H A R D S.
2.2 *5 25 *4 , 2.2 5 20 **
2.6 15 10 ** 2.6 ** ** **
2.8 19 6 ** 2.8 20 5 **
3.0 20 5 ** 3.0 21 4 4*
3.2 21 4 ** 3.2 22 3 **
3.4 22 3 ** 3.4 23 2 **
3.8 23.5 1.5 on 3.8 23.5 1.5 a*
402 2405 05 *5 402 2405 05 3*
5.0 25 ** ** 5.0 85 ** 4*
5.0 85 ** ** 5.0 25 ** **
5.4 24.75 ** .25 5.4 0* ** as
5.6 24.5 ** .50 5.6 ** ** *e
5.8 24 ** 1.0 5.8 24.5 ** .5
6.4 22.5 ** 2.5 6.4 24 ** 1.0
6.6 21.5 ** 3.5 6.6 23.5 ** 1.5
6.8 20.5 ** 4.5 6.8 23 ** 2.0
7.0 19.50 ** 5.5 7.0 22.5 ** .2.5
702 18.5 *G 605 702' 22 5* 5.0 .
704 1706 ** 7.5 704 2105 ** 3.5 i
706 1700 ** 8.0 706 21 *fi 400 .
708 1605 Q* 805 708 2005 §* 4.5
8.0 16.0 ** 9.0 8.0 20 ** 5.0
8.4 r 15.5 V ** 9.5 8.4 19 ** 6.0 .

 

 

 

 

 

 

 

 

were sterilized in the dry air sterilizer while the medium, the

water and the acid or alkali were autoclaved separately. The

proper amounts were mixed in a thoroughly steamed inoculation :
chamber an pipetted into the tubes and the preparation dishes 1
by means of sterile pipettes provided with cotton filter plugs.

A suspension of spores was used as inoculum in the case of the

i
E
parent strains of Colletotrichum lindemuthianum and Cladosporium \
fulvum while in the case of the variants of both fungi a bit of

mycelium was used.

 

 

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

v
. . . . . . .
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. Changes in uninoculated media after 1-1/2 months.

_c_o_ons’ medium + Indicator

 

 

Richard's medium + Indicator

art Final Indicator Start Final

2.4 2.4 T.B. 2. 2

2.4 2.4 " " 2.

2.8 2.8 9.3. 2.8

2.8 2.8

3.2 3.2 BPB 5.7

3.2 3.2 " 3.7 3.7

3.8 3.8 4.8 4.8

5.8 4.8 4.8

4.2 5.0 5.0

4.2 5.0 5.0

5.0 5.0 MB 5.8

5.0 5.8 5.6 BC?

6.6 Faded MR 6.4

5.6 Faded MB 6.4

6.4 6.4 BTB 7.0 6.4. BTB

6.4 7.0 7.0 BTB

7.0 7.6

7.0 7.5 3.1061" °X° §§i§°§§m PR

rem-“e.
8.4
8.4 6.6 BTB
8,4 8.4 TB
8.4 T

 

 

 

 

 

 

 

 

\ .ItlullIllII'u'ir \i I
. .1...

Pi Inw’u‘

The cultures were grown in the dark. About 12 days after
inoculation thehcolor and the relative amounts of growth were
observed. Tables XII, XIII and XIV summarize these observations.
No observations were made at that time on the black strain
of gelletotrichum lindemuthianum.. The variant however, respond-
ed in an interesting way. Its growth range was between pH 3.2
and 8.4. It grew true to type in the series on Richards medium
producing a slimy growth at the beginning, of creamy or white
color, from which white aerial mycelial tufts were given off
a little later. On Goons’medium, however, it seemed to revert
back to the black form. It would have been interesting to
study this particular point further and especially to deter-
mine whether the black color was due to fruiting or not.
Oladosporium fulvum responded much more readily. The parent
form grew well on the media of pH 2.819 7.6 producing the
characteristic dark olive green color of the submerged growth
which is produced on nearly all media at the base of the slant
and the growth under the agar and also the much lighter yellowb
ish olive color, characteristic of the aerial growth. A
purplish color (Vinaceoue lilac, daphne red) was produced towards
the alkaline side of the series and this color is characteristic-
ally produced on certain media like Cat and Corn Meal Agar and
Prune Juice Agar. The same purplish color was observed in the
Variant strain also towards the alkaline side. There was a
greater variation in the color of the aerial growth on the
Richards series, but there was no characteristic distribution,
the same color being found on both the acid and the alkaline
side. No purple tints were found on ;ither the parent or the
variant. On the contrary the buff color that tinges the mycelu

lo 10‘ lulll tillyfl.

 

 

in

 

link-3!}: Lula ... A! at: .%.E{EEH7F

I.
rl‘lle'n.. '5: III .7 .. u I ‘u w y\ \III‘ ,

 
 

 

 

-5qi

.um of the varianv‘towards the acid side was present throughout the
teries on this medium. In general, as has been observed by many
nvestigators, fungi tend to shift the reaction of an alkaline
substratum towards the acid side and this was the case in the
experiments Just described. No further observations were made by
3119 writer on this experiment, but after l-l/Z months final notes
tare taken by Dr. Coons and these have been incorporated in the
;ables.

It will be seen that in the case or both Species the range of
tolerance toward acid and alkaline conditions is practically the
lame. Ihe effect of the parent and variant strain upon the medium,
taking into account the relative growth, is alike. The distinctions
in color are maintained; and what is perhaps the most significant,
in spite of the great variety of growth conditions presented, there
is a complete absence of sporulation in the variant as contrasted
Iith heavy spore production in the parent forms in some cultures.

The differences of the two forms manifested in the previous :

work seems to be largely maintained.

 

.03. .

......

 

.. ...”..weemtww 32);}..{lp‘m :5 Ambit.

a ... \v. 1.1-x... up... ......D‘.J.s..w.fi 5..“ .7 ..I . ...!

 

desperison of the two strains or

medium of varying H-ion concentration:

  

 

rat Form

  

 

-53-

mm in Coons’
ultures in test tubes.

Variant Form.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

"111130 1818116: H 4 white islands; 1‘ 2 white islands.

Namath color afterJPfi PH Initial Growth c616:- after PH PH
after 1-1/2 me. after after PH after @1-1/2 mo. after after
1-1/2 12 da. 1-1/2 . 1-1/2 12 daJ-l/l
Ge no. mo. - mo.
, (Merely 2.4 2.4 2.4 a (liarer 8.4 2.4

starting) starting)

. * 3' “ 2.4 ' 2.4 :I: " 8.4

77. ]Olive gray 2.8 2.6 2.8 + Bluish gray 3.0
: 81'0“!

' + II It ' 2.8 + ” ' 3.4
.. |
‘ ++ Warn but! 3.2 3.2 «H Light buff 3.2 3.4

! +4. I. u 302 ++ ' ' 3.4

.L A '- s * ~~

1 .+. Tawny 3.6 4.0 3.8 +. may. but! 15.6 4
+§+ " 400 ++ 17 VI 4‘2
.... Buckthbrn 4.2 ‘ 4.6 4.2 ... Dark(3ub- 4.2 4.6

Brown . m ed
++++ Buckthorn 4.4 +++ ‘18 ‘ 405

L: v Brown fl - w _;
++++ Buokthorn 5.0 5.2 5.0 ++ Light buff 5.0 4.8
_ Brown
“... Buckthorn# 5.2 .. " " 4.3

grown g — t _
+++ Ruckthorn 5.6 5.6 5.6 +++ Light buff 5.6 5.4
a Brown " _ _
'+++ 5.6 +++ " " 5.4
4 - _i
; ++ Buckthornf 6.4 6.2 6.4 +4» Light buff 6.4 6.2
F Brown
F ++ n n 6.4 ++ " n 608
“t . w— w Tb A n
, 7.0
_jL . - n n
«H Buokthorn 7.2 7. 7.6 «H Light tan 7,2 7.2
Brown

++ " a 7.2

+ i huutw f 704 €04 ++ Pinkiah 804 608

,. vinaceous white ‘
22::— ;_-:-JL—-—--“-——-——- in. H fin a $492.1

 

 

1.7%.»... ... ..

 

 

, Comparison of the two strains of C
Richard's medium of varying H-ion concentration:
in test-tubes.

rerant Farm

AL

-59-

331122.011
Cultures

Variant Far m._

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

el Growth Color E PH Initial Growth Color E PH
after fter after PH after iter after
1-1/2 n2 da. 1-1/2 1-1/2 2 da. 1-1/2
‘1 .90 no. mac m0.
0 2.0 8.0 O 2.0
O 2.0 O 2.0
+ [Light 2.8 2.5 2.8 + (Sub-
iOlive merged)2.8 2.5
‘Gray
+ ' " 2.8 2.5 . Light
; Gull
J Grqzi 205
1' Gray 3.5 302 507
‘I‘ Light 305 204 1? P310 306 208
. olive , Olive
M. are; i 9’31 i
++ T68 406 4.8 ++ Ivory 406 298
green yellow
++ " 4.6 2.7 ++ Light 2.8
grayidb
olive
+ bunera 4.8 2.6 5.0 ++ Olive 4.2 2.6
Gray ‘ . buff
+ " 4.8 ++ Vina- 2.4
scene
but!
73+‘fi’ Tea 5.: 2.3 558 +++ (Ochf- '7‘3
green aeous 5.4 2.8
tawny
++ " " 5.4 I 2.6 +++ 7 1 2.4
4"? 0117. 506 604 ++++ Gray '02
lake
++ I I 8.4 20‘
++ Light 606 3.2 700 ++ P810 606 204
Olive olive
gray buff
++ Olive 6.6 2.6 ++ " " 2.5
8231—, a
'7"? Olive 700 206 706 +++ Pinki 700 2.4
gray buff
++ Olive 7.0 2.6 +++ " 2.4
_g are
++ Olive 4.1 8.4 + Pinki h 4.4
. gray but!
___ ++ Olivemfi 2.5 + " 3.5

J

......

VflwPF

 

114!

 

 

 

 

- e a
o g n n I u
a I e
o a
n o x o u n
5..
5“ If 1..; on
‘4}?

 

 

2 ~60-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

comparison of the two strains of on Coons
andR 2ichard's media of varying H-ion cmcentration: cultures
in preparation dishes with filter paper cones.
Parent Fotrm Richard' 3 Medium Varianflogm.
- rwth Color Spores 110. PH Growth Color pores
- 3 ..- -
. 2.
‘ - 5: - -
" ontam. ll 2.8 + Greenish -
4» Dark Gray- ‘ g 12 + " ..
f . green - A
1: ontam. l9 5 7 Contam. .
" ++ Dark Gray- 4» 20 ’ + Tan __
A green .
+ Blue green - 27 4 8 ... Ian __
’ ++ Greenish "' 28 ' + " _
‘ +fi Olive green I - 35 5 O dontam.
+ w w - 35 ° w
+ Olive green + 43 5.8. + Pale vina-’ __ #
. . . ceous lilac
+ Greenish black 44 + ,. __
Olive green + 51 5 4 ++ Bufl _-
" " + 62 ° Contam.
" Olive green 7.0 ++ 3W _
60 «H " -
Blue green + 7 5 ontam.
" " + 58 ‘ Contam.
Blue green + 75 ++++ Park gray ..
i _l. 76 8'4 L ++++ " " ...
Coons‘ Medium

    
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

It 83 2.4 ++++ 9108111811 _-
Grayish 84 +++* '
'w + aflc greenish + 21 ++++ Grayish _
Ld ‘ . ~ 2.8 black
+ Dark Green + 92 ++++ " " -
++++ fight hm— + 99 ++4+ ay'ish _
504 ‘ ' - 3.2 bDCk
++++ " " lOO ++++ " " __
a +++ Dark purple + 0 ++ Light vina- _.
5. . 3.8 , ceous lilac
+++ " " ‘ + 108 +4» Light Vina- _._
g L . . ceous lilac
“'— +++ ark purple + ++ Light vina- _
4.? . 4.2 ceous lilac
. +++ " " x + 16 + White, _.
E +++ " + ++e Pale vinaoeo s _
500 +++ W I + 24 500 +++ 7 I I —
I; +++ + 5 6 +++ _ _.
' +++ " " + 32 ‘ Contam.
par—file + ++++ Light vinacebus .-
,, lilac, buf‘f to
6.4
i ‘ 0 4:? n n. 'w ‘-

 

 

 

 

 

 

 

 

 

 

 

ant inued .

 

-608!

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ngqumghd‘nméflnnti \
jfimwth Color Bpores 0. PH browth Color Spores.
++++ Purplish 147 ++++ esp vinaoeous _
1 . brown + 7,0 . lavender
+4... " " + 148 Contam.
++++ “ " + 155 «H Depp vinaoeous _
titu- " " + __ 156 ++ " " " _
I + ' pufilsh -- 8 ++ v _—
«l! " xxe + 164 ‘ Contam '
:-White Island.

-61...

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Comparison of the two strains of 0.2119111211211—

MW II when grown on Coons’ medium of

varying concentrations: Cultures in test tubes.
fiParent Form ~ Variant Form .

L Relative Color cervuli pH Initial Relative Color Acervuli pH
growth ith after pH growth with after
(after * ores 1-1/2 after . spores 1-1/2
‘1-1/2 mo. 1-1/2 mo.
.3. W mg. f

O 204 O 204

'44 . o itef I 3.2

Black 2.8 ’
+++ ++ffi7 2.8 + _fif A 3.2
+++ Black - ... 3.6 3 2 + it? 3.5
+++ " 4+4 3.6 + ite# o 3:5fi
+++ " +++ 4.4 .

, 3.8
4:... ++. 4.6 ++ ite# o 4.6
+++ +++ 4.6 ++ ‘ its 0 4.6

_ , _ 4.2 .. 3
#1 " g++ 4.6 ++ ‘ itef o 4:6w
+++ " +++ 5.4 5 O + } itsf o 4.4
o 5.0 +++ 2 ltd; L o 5.2
5.5 5 5 + Eu. 0 4.2
lack +++ g 6.6 4+. 1t6# o 5.4
" t++ 6.2 6.. ++ lghite# o 6.
"_fi +++ 602

7.0

7.6

++t Black +++ 7.63» 8.4
... +++ 7.2 ' ++ _iwhitoi‘ o 7.2

 

 

 

 

 

 

 

 

 

 

urged.

 

 

1L

.4 (....
K 11.!
-..LHLxs -

 

Comparison of the two strains of

Gollaini_i_hun
Wuhan grown on Richard‘s medium of
varying H-ion contrations: cultures in test tubes.

r 0

-62-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

_w Parent Form‘ , - fl Neriantli‘orm a
:21 Relative Color cervuli pH Initial RelativeTColor cervuli 11
growth ith after FE growth ith i't r
after spores l-l/ 2 after ores -l72
1-1/2 mo. 1-1/2 0.
._.-LEE" J m0. 1
o
4 2. 2 °
fi 0 0
Not 0 ‘ 2.6 o 2.8
record- 2'3
d .
v 0 % 206+ ‘I’ O 208
+++ lCream + {4.4 3 7 ++ White 0 4.4
... ° + '5.2 4+ , o 5.1
++ ite# . o 4.2 4.2 ~
... 32.11 .— 5.7 +++ White# 0 5.2
ink
++ its? . 4.2 5 0 ++ White# 0 4.6
+++ 311011 +++ 6.6 +++ " O 5.5
in]:
+++ hell ++ 6.4- ++ White 0 4.6
+++ Shell +++ 6.4
3 pink
+++ Shell +++ i6.3 6.4 +4» "hi-1307‘; o 5.4
? pink b
black V) th
acervul
+++ " " " +++ 6.3 ++ " 0 530
H II n I
. 4++ +++ 6.2 7.0 4+ 0 5.8
__g +++ " " " +++ 6.4 +++ " o 4.9
+4» Shell ' + 509
g ++ " " o 6.0 H» White 0 5.8
4+ Shell 5,5 ‘ " o 6.5
pin]: +++ 8.4 +
__ ++ -" " +++ 6.2 + " 0 5.2

 

 

oergad .

 

14 v 117M}...

 

 

 

-68.-

Comparison of two strains or C 112 otrichum
ianum II,‘whsn grown on Richer

varying.H-ion concentration:
dishes with filter paper cones.

_Parent Form

A

Richard's sol.

s and

linde uth-
eons media of

Cultures in preparation

Vari ant Form

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

rowth Color pores pH: Growth. Color _Spores
20 "'

+++ lack + 2.8 -

lg; " + -

++++ ' + 3.7 Contaan

133+ ' + "

,+++ - + Canteen

.- 4.8

+145; " + + _v

Gentam. 5. Contam.

“H 3120]: + "

++++ " + 5.8 Canton.

.6...” 0W ' + f Cram! -—

++++ Black - ++++ Cream. ._
. 6.4

p114 " - Contan.

e+++ ' - Contam.

Contafl. 70 ++++ cram -

++++ " + 7. 6 +++ Cream -

gi++ ' + +++ ' -
I

++++ + 8.4 +++ Cream -

gontaui- +++ " _

Coons Synthetic Sol.

r- - 2.4 ' + Cream. -

ggntamo + L _-

1+ Greenish - 2 8 ++++ Dark -—

black ' gray
r+++ " " + ++++ " " -
++++ Black + ++++ Crayi 8h -
3.2 black
y+++ w + ++++ 3013 black -
in places

43“ + + Jet black -

.+++ + 3.8 + + in places

.+++ ” + ++++ " " -

 

 

u ..9‘

-62b-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Continued)
Coons Synthetic Sol.(Continued)
tfiwrm , Variant Form
th Color Spores pH Growth Color Spores.
Black + 4 2 +++ Red brown -
" 4 ++++ Black -
" 5. 4+ Black -
" + ++ " -
" n + 506 ++ " o
" + ++ " -
Black + Black -
+ "6.4
" + Contam.
I + 7 4.4-... 3180K '
u + . +++ " -
I u _
+ 7.6 +++
" ... , Contam.
" Black -
H 8.4 fl
_ " ++ ++ " -

 

 

-63-

ch or TEMPERATURE AND REACTION ON 9930;; AND GROWTH.

In connection with another experiment carried on in
JBerkeley, Calif. both parent and variant strains of Glade-
npcrium fulvum_were grown on plated Shive's Best Dextrose agar
(79) varrying in pH from 4—8 and kept at 30-320 0.. 20° 0., and
10° 0. The medium was prepared in lots of one liter, brought
to the desired Hyion concentration by the addition of acid or
alkali, tuhed at the rate of exactly 10 cc. per tube and steamed
for one hour on three consecutive days. Duplicate plates were
poured, allowed to cool and inoculated in the center. They were
wrapped in paper and kept at a constant temperature as mentioned
above. At intervals the plates were examined for color and

rate of growth. The composition or this medium is as follows:

MgSO4 2.12 gms.
KH2P04 1.36 '

. Reno3 5% 1 drop.
Dextrose 20 gme-
Water . 1000 cc;

The following table gives the number of cc. of l/B KBH re—
quired to adjust one liter of the medium to a given pH, as
determined by Sideris (79).

TABLE XVI. .
cc. of 1/5 KOH required to adjust 1 Liter of Shive's
Best Dextrose Agar to a giveopfi.

‘00 P 405 n 500 I 502} 504 b 506 l 5.8 D 6. 0L 6. 2 c

 

 

0°. l/B **Q t 04 H 08 I 100 L 108 p 206 D 500
K03.

 

7000p 702 b 704 r 706 d 708

 

pH i 604 L 606 1 608

 

 

 

 

 

 

 

OEéEl/b b 902 “1105 11405 }1705 $2107 “2509 03008 13307 }560 5 a

 

 

 

I

 

 

 

.64-
TABLE XVII.

Effect of Temperature and reaction on Color and Growth
of the Parent and Variant Strains of ados ri :nlxun
grown on modified Shive'e Best ledium of varying pH and
kept at different temperatures.

 

Temperature 30-32° 0.
No growth beyond the point of inoculation at which a slight
weak growth was observed 54 days after inoculation.

 

Temperature 10° 0.
lo growth whatever.

 

Temperature 20-25° 0.
Dian. AFTER 3 DAYS Diem. AFTER 8 DAYS
pH 4.0 m. p m.
Par. 3.0 Slight growth, whitish. 6.5 Surf. Light olive green
F Under.led Brown.

 

War- 2.5 White. 5.5 Surf. White
Under.Yellow Broggi, j
il‘Sis’ “"1
Par. 1.5 Greenish Brown 4.5 Surf. Light Olive Green 9
Under.Green P
Var. 2.0 White. 4.0 Surf. White i

I
!
Under.Dark Yellow Brown H

 

 

 

pl 6.0
Par. 2.0 Greenish Brown. 3.0 Surf. Light Olive green
Under.Green
Var. 3.0 White 4.5 Surf. White
Under.Dark Yellow brown
pH 7.0
Par. 2.0 Greenish brown. 6.0 Surf. Light Olive green
Under.Purple brown
Var. 2.5 White 4.5 Surf. White
. . Under-Orange Brown
I
pH 8.0 . i
Par. 2.6 Greenish Brown 5.6 Surf. Light Olive green
Under.Purple, white edge.
Var. 3.0 White 5.0 Surf. White

Under.Purp1e.-

 

 

 

 

 

I r .11 .' _‘n (I

 

 

 

XVII. (Continued)

 

AFTER.54'DAYS

Surf. Brownish Olive 30

Under. Dull Greenish Black 47

S. White center, clay color edge.
U. Hessian Brown center, Ochraceous 0range edge

v

 

Light Brownish cliwe 30
‘I. Dull greenish Black 47
S. Pale Yellow Orange

Us nae]. 1‘s

 

 

Light Brownish Olive 30

U. Dull greenish black 47

S. White center, Oohraceous buff edge.
U. 3‘20]. 1‘.

 

8. Light Brownish Olive 30

U. Dull greenish Black 47

s. White-Light Ochraceous Buff.
U. Hessian Brown.

 

 

8. Light Brownish Olive 30

U. Olivaceous Greenish Black 47
8. White-Pale ochraceous Buff.
U. Hessian Brown.

 

 

 

 

.304)”. El.» u.”

 

-53-
Table 3711 gives the growth of the fungus lDiameter of solo-
nies in m.m.) and color of the aerial and submerged part,
three, eight and fifty four days after inocultion at the three
temperatures used.

lakemson (54) found that the temperature limits for best
growth.of this fungus were rather narrow, the fungus growing
best at betwwen 20 and 26° 0. The experiment reported above
‘ and the fact that the writer experienced great difficulty in
keeping this fungus in culture during the summer in Fresno,
Oalif. (Av. temp. 32° 0.), confirm these results. Temperature
therefore, did not enter as a factor in this experiment. The
variant was found to be a more active grower than the parent
strain as indicated by the colony measurements and both parent
and variant seemed to grow rather uniformely within the Ehion
concentrations limits of the experiment.

Regarding the color, this experiment confirms the results
of the previous experiments on the effect of the reaction on
the medium. No striking variations were seen even between
the extreme ends of the series, in either the variant or the
parent except perhaps the development of the purple color
in the variant towards the alkaline and as had also been
observed in previous experiments.

A repetition of this experiment gave similar results.

 

I’ll}- ! I

 

-57-
PATHOGENIOITI.

The majority of the variations induced through unfavorb
able environmental conditions have been among the saprophytic
~ fungi and bacteria. Of the spontaneous variations or mutations
reported in pathogenic fungi a few do not show reduced pathogenic
action while in others the reverse is true and in still others
data are lacking.

Edgerton (36) found that the Glcmerella mutant reported
by him grew very slowly on apple. The Brachysporium mutant
reported by Bonar (12) was not pathogenic on white clover and
came from a parent of reduced virulence due to artificial
cultivation for many generations. Bennett, who according to
Ooons (25) investigated white forms developed from a culture
of Phcma apiicola found that they are also attenuated forms.

On the other hand Orabill's Coniothyrium variant (31) was
pathogenic as also were the Glomerella variant reported by Dastur
(32) and the Botrytis reported by Brierley (15). Definite
information is lacking in the cases reported by Burger (19),

and Stevens (83).

Pathogenicity experiments were carried out using both
Parents and variants of Oladosporium fulvum and ficlletotrichum
lindemuthianum. Potted plants of Stone and Beauty Varieties
of Tomatoes and Rust Proof wax Bean were inoculated in the
following way. A vigourously growing young culture of each
fungus to be tested was macerated in a mortar under aseptic
conditions, sterile water added and filtered. Half of the
filtrate, a drop of which was examined microscopically for

presence of spores or mycelium, was sprayed in each case on

 

 

 

_edu
five plants, the other half being used in swabbing the under
side of leaves of wnother set of potted plants. A third set
of five plants served as checks. The inoculations were made in
the evening, and small flecks of sterile absorbent cotton were
placed on the inoculated leaves to prevent rapid evaporation.
Each set of plants was kept in a separate moist chamber in the
greenhouse until the next morning and were then brought into
the greenhouse.

These experiments were all unsuccessful, not a single case
of leaf mold or anthracnose being noted on the inoculated
plants. This failure of the inoculations was probably due to
high temperatures and the heavy white fly infestation of the
greenhouse.

Oladoepcrium fulvum. The experiments were repeated in 1924
in Berkeley, Calif- Pour potted plants of the Stone Variety of
Tomatoes were used in these experiments. Two of them were
inoculated with the parent strain, and the other two with the
variant strain of Oladosporium fulvum, the inoculations being
made in separate rooms of the laboratory under the usual aseptic
conditions. A drop of sterile water placed on the lower or upper
surface of a leaflet, was inoculated with a little mycelium
from a young vigorous culture and covered with a flock of
sterile cotton. Each plant was put then under a large glass
bell-jar for five days and kept exposed to light near a north
window of the laboratory at 20-26° C. After this period the
plants were gradually brought into the Greenhouse. The experi-
ment was exactly repeated using four potted plants of the Beauty
Tomato Variety.

These inoculations were entirely successful. Both the

 

 

-59-

parent and the variant produced spots at every point of ino-
culation, more that thirty spots being counted in each case.

No spots were seen at other than the inoculation points and

and although these plants together with many control plants
were kept in the same greenhouse there was no natural inflection.
The spots produced by the parent strain were all characteristic
of the disease as described by Makemson (54). There was an
olivaceous growth on the under side of the leaf with an
abundance of spores characteristic of the fungus. The upper
surface of the leaf in the infected area turned yellow and
became dry. The spot spread irregularly from the point of
inoculation..

The spots produced by the variant strain were elliptical
well defined in appearance, rather dry with yellowish flakes
on the surface. There was no mycelial growth on the under side
of the leaf. Free hand sections through the infected portion
of the leaf showed the presence of mycelium throughout the
leaf tissue. Five plates were poured from leaves infected by
the variant using Ozapeck Dextrose Agar for Medium. In every
case the white form of Cladosporium grew on these plates.
Similarly infected leaves were placed in moist chambers. The
white variant grew from the margin of every spot. Plates from
tissue infected by the parent strain showed an abundance of
growth of the typical Oladosporium fulvum.

Plate 12 shows leaflets of Tomato artificially {footed with
bOth forms or Oladosporium. Isolations were made from the part
of the leaf infected both by the parent and the variant strains

and reinoculations were made on potted Beauty Tomato plants in

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o-69—

parent and the variant produced spots at every point of ino-
culation, more that thirty spots being counted in each case.

No spots were seen at other than the inoculation points and

and although these plants tagether with many control plants
were kept in the same greenhouse there was no natural inflection.
The spots produced by the parent strain were all characteristic
of the disease as described by Makemson (54). There was an
olivaceous growth on the under side of the leaf with an
abundance of spores characteristic of the fungus. The upper
surface of the leaf in the infected area turned yellow and
became dry. The spot spread irregularly from the point of
inoculation.“

The spots produced by the variant strain were elliptical
well defined in appearance, rather dry with yellowish flakes
on the surface. There was no mycelial growth on the under side
of the leaf. Free hand sections through the infected portion
of the leaf showed the presence of mycelium throughout the
leaf tissue. Five plates were poured from leaves infected by
the variant using Ozapeck Dextrose Agar for Medium. In every
case the white form of Cladosporium grew on these plates.
Similarly infected leaves were placed in moist chambers. The
white variant grew from the margin of every spot. Plates from
tissue infected by the parent strain showed an abundance of
growth of the typical Oladogporium fulvum.

Plate 12 shows leaflets of Tomato artificially {Tected with
bOth forms 0! Oladosporium. Isolations were made from the part
of the leaf infected both by the parent and the variant strains

and reinoculations were made on potted Beauty Tomato plants in

 

 

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~70-

bloom. The methods used were those described above, except that
inoculations were made on both the upper and lower sides of

the leaflet. Ho spots were produced from upper surface inocu-
lations. Typical spots similar to those described above
developed on all (fifteen in number) of the lower surface
inoculations- Inoculations on the calyx and stem of flowers
produced characteristic spots. Small tufts of white mycelium
were found at the margin of the spots produced by the variant-
Such tufts were examined under the microscope for spores.There
were no typical spores except a few spore like bodies believed to
be chlamydospores. The mycelium was thin, densely branched,

and purple in color.

Dastur (32) found that passing the variant form of Glomerella
through its host restored its alost sporeyproducing power, but
this was not evidently the case with Qiggggpgziym tyizyg, since
it did not produce spores even after the second passing through
its host. From the experiments described above it becomes
evident that both the parent and variant are parasitic on the
tomato plant, but growth on the host aiqhgyteg39fgst properties
of color and fruiting of the variant. Boner (12) working with
Brachysporium which had lost its pigment but not the power of
fruiting found that the variant was not parasitic and the
parent had lost the greater part of its power of parasitism
through prolonged cultivation on artificial media. This is not
the case with the parent form of Cladosporium fulvum, which
infected every leaf inoculated although it had been under

artificial conditions for over four years.

 

to

 

 

Oolletotrichumfilindemuthianum; Golden Wax Bean plants were ino-

culated with the parent and variant form of Colletotrichum

 

lindemuthianum Exp. Sta. Strain II, using spores or bite of

 

mycelium in the same manner as in the tomato plant inoculations.
Inoculations were made on both the upper and the lower surface
of the leaves. No spores were formed on any of the upper
surface inoculations or on the control leaves. The results were
not so definite as in the case of Cladosporium. The leaflets
inoculated with the white form of Colletotrichum turned yellow
but no definite spots developed except in one case where typical
anthracnose spots were observed. No spores were found. The
leaflets inoculated with the black form of Bolletotrichum
showed typical spots of anthracnose with an abundance of spores.
*fiflflflfi-fi-flfiflfifl-fl-flflfi
D I S C U S S I O N A N D C O N C L U S I O N.
9! 4! fl 4} 1*

As mentioned at the beginning of this paper, the proposed
experimentation, that was intended to ppocure the arguments for
a discussion of the whole problem, was not entirely carried out.
Such experiments as are recorded here, preliminary in their
nature as they are, do not throw light on this complicated and
much discussed problem. Perhaps it would be of interest to give
here an account of further experiments that the writer had in
mind for the same reasons that the present experiments are re-
corded - to form the basis of further work.

These experiments are outlined chiefly with the fungus
Oladosporium fulvum in mind. They could be varied to apply to
the other fungi under investigation. And first, a fundamental

study of the nutrition of the present fungus is necessary to

 

II-I UIIIII .

 

 

 

 

furnish the basis for nutritional work. Nutrition is credited
by De Vries as the cause of all modal or environmental modi-
fications which are deviations of varying magnitude from the mean
and have been found to yield to selection in changing the mean
of a specific character. Brierley in 1918 (14) finds also that
modal variations are directly and constantly induced by parti-
cular substrate. The present investigations show that the strain
of Oladosporium fulvum used in these experiments is in the state
of mutability according to De Vries. since the white variant

has been given out frequently irrespective of conditions of
growth. For the nutritional experiments a strain should be found
not in a mutating state and this should be compared with the
present strain and an attempt made to induce mutability in it.
Further experiments along this line would be called for by the
pregrese of the work. Besides nutrition other environmental
factors should be investigated in order to corraborateevidence
presented by other investigators working with other fungi. The
effect of poisons, acids, alkaloids, metabolic products etc.,
the effect of hydrogen ion concentration kept constant by
frequent adjustments throughout the course of the experiment,
the effect of light, temperature, controlled humidity should

be carefully studied. These should be studied in order in l.

to induce mutability in an otherwise non-mutating strain, 2.

to find out if any of these factors regulate the appearance

of variants in an ordinarily mutating strain, 3. to determine
their effect in changing the mean of any specific character.

In the case under investigation a study into the pigmentation

of Oladosporium fulvum is necessary.

From the experiments in hand one conclusion could be drawn
safely. The environmental modifications tried, exercise an

 

 

 

...‘ui.

41 \J.-Or1....a .«1 HJl In: 5:44 ...): «.1 . a... u.

.. iqflttufdl .
r

 

 

effect on the color and fruiting of l dos orium fulvum,

they cannot, however, induce the type of variations ordinarily
given out at unprOgnostigated and irregular intervals by the
strain under investigation.

One cannot but agree perfectly with De Vries (87) that
”nothing is more variable than the meaning of the word variability"
(1.0. p. 43) and perhaps the word mutation be substituted for
variability. At the beginning of this paper a series of inter-
pretations of the meaning of the term by various authors and
by the formulator of the whole theory was given. This list can
be extended, but without any particular benefit. The confusion
has led several writers to avoid altogether the use of the term.
Horishima (58) in 1921 reporting several cases of adaptive changes
in bacteria, STtEEEOsus in particular, thinks that the term
mutation should be used only with higher plants and should not
be introduced into bacteriology 'for the bacteriolOgist,
who studies his species not only from the morphological point
of view, but also with regard to biochemical and immunological
reactions, and who observes not a few generations only, but
hundreds and thoudands of generations, would almost surely
have to modify the conception of the term in such a manner as
to cause confusion to the botanist. It, therefore, seems
advisable to leave the term mutation to the botanist and, for
the present at least, to speak of atypical varieties of bacte-
ria or simply variants.“ The present writer has also avoided
the term mutation for similar reasons. Stevens (83) uses the
term saltaticn with a new meaning to cover variations in non—
sexual generations of fungi. Chaudhuri (21) followes his

example for the same reason.

 

 

~74~

‘lrterley (15) strongly objects to the use of the term
mutation 'It is not the possibility of mutation in the fungi
(and bacteria) that is here denied but rather the compulsion
‘to accept that interpretation of the evidence as it now stands“.
‘He rejects the use of the term on the grounds that there is no
assurance of genetic purity in asexual fungi. Jennings (45)
in.l911, Blakeslee (8) in 1920, Shear and Wood (76) in 1913,
Brderley (15) in 1920, and Stevens (83) in 1922 agree that in
single spore cultures of asexual fungi we deal with pure line
individuals. The term mutation has been applied by De Vries
to germinal vaéations, arising idependently of environment;
remaining constant afterwards, in a rarely self-fertilized plant
grown from seed, the genetic purity of which.has been seriously
attacked by Bateson and other geneticists and proven to be
a hybrid whose segregation is checked by balanced lethals as
it has been recently explained by Bradley Davis (33). The
modern use of the term, however, presupposes according to
Brierley (15) and Davis (33) an absolute certainty of genetic
purity. In the homozygous asexual generations of fungi coming
from single spores and carried in culture for countless gene-
rations there is scarcely any chance cf genetic contamination.
Anastomosing in single spore cultures cannot be accounted as
destroying the genetic purity. Shear and Wood (76) discussing
the question of 51-321: heredity and variation in Glomerella
say 'there is no reason to believe that the mendelian theory
is involved or heterozygosis takes place as there is no union
betwwen nuclei of different individuals and species. If there
is any union between cells or nuclei of a single spore organism
there is nothing new coming out of the union, no new characters

transmitted,,,,..it is hard to see how two closely related

tn

nuclei in the same individual or cell could add essential
characters to the organism. Individuals from single spores
mmet be considered homozygous...“ ”Whatever differences the
progeny of such individuals might show would evidently be due
to Mutation or some other internal cause, such as the renewed
expression of latent hereditary characters.'

Brierley (l5) rejects the pcssibilty of a mutatiwe
origin of variants in the fungus studied by him on the grounds
of contaminaticns by anastomosis in some remote time in the
life of the strain before its artificial cultivation. Although
conceivable, it is hard to believe of an interspecific or
other anastomosis in Cladosporium or other fungi that would
so modify the nuclei of a cell and build up a system of
balanced lethals covering suchp modification for many genera-
tions, finally breaking up repeatedly into the variation
Observed. It would particularly be hard to thing of such a
happening in the variation recorded in this paper, via. the
loss of a single character, that of fruiting, which apparently
is accompanied by the loss of the olive-brown pigment character-
It is more probableto think of such a case as a retrOgressive
mutation (mutation by loss) or class it with the iegetative
mariatinns known as sports or bud variations which De Vries
also classes with Mutations. The doubt of a genetic contamination
by anastomosis fades before the doubts of genetic purity in any
pure line of higher plants where contamination by cross ferti-
lization and hybridization is much more probable than contami-
nation by anastomosis, in non-sexual plants.

Of the variations in fungi reported in literature the one

 

 

 

 

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neakly ‘ .
Inost ‘ approaching the one described in this paper is the

'whlte non-fruiting variation reported by Crabill (31) in 1915
in.Ooniothqrium pyrinum Sheld. This fungus is pathogenic on apple
the white strain being also pathogenic. The loss of the olive-
brown color is accompanied by a loss of fruiting. It has been
Obtained several times and has been constant. Environmental
modifications could not induce its throwing off. All these
correspond with the variation of gladgfipgninmutnlzum_and
Collgtotrighgg lindemuthiangm reported in this paper.

.fifl-Qflfi-Q##fl-flfifi‘fififl'flfi-flflflflflflflfi

S U M M A R‘Y .
assess

Albino forms with a loss of fruiting are reported in
Oladospgrium fulvug and Colletotrichum lindemithianum;_

A comparison of the growth of these forms along with
their parents on different standard media, and microscopi-
cally is made.

The parents and variants are grown cn_a series of media
arranged according to the triangular system for the purpose
of studying the effect of nutrients on color production and
variation. Color was found to vary in ngdosporium fulvum
under the conditions of the experiment. 2H; other fungi used
did not respond.

Light was found to have no effect on variability and color
of Oladcsporium fulvum.

Both parents and variants of_g. fulvum and_g, lindemuthia-
nun were grown on Coons‘and Richards liquid media ranging in
pH from 2:8.4 in order to study the effect of pH on variability
and color. Both strains of the two fungi were found having a

 

 

 

 

 

 

 

n77-
wide range of growth and varied somewhat in color under the
conditions.
The temperature limits of both the parent and the variant
of Clad._£ulvum are found betwwen 20 and 25° 0. No striking

 

‘variations in color were observed on growing this fangus
on solid media varying in pH from i=8.
Both the parent and the variant of_glad. fulvum and Coll.

 

lindemuthianum_were found to be virulent parasites on their
respective hosts; passing through the host did not restore
the lost characters of the variants.

A review of literature and a discussion of the possibility

of mutations in asexual fungi is given.

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Chestnut bliffit fifivus ard its naur rilatives.
U.3.B.A., B.P.I.., Ciro. 131, 1913.
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developrent Ct tie 917k rest pet disease of Qatar;

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with observaticrs on the morphology of helrintbcev
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Q2 ‘Q'fi ‘ -_‘1" V ' "f(1 {'1
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‘7EJT’ P‘T & J.O.HALL. lCOC. VariatiChs of fury i Cue to

04—10

envirhrrent. sot. Gaz. 43:47:71. lCOC

q: C‘ r ‘ ‘Y‘V . . ' _ ,, T1 - . ..r“ . —‘ o 1
“° “J -==:':LRE, F.C. & :...flflluilss. 1003. bporntrlcnum bud—rct Cf
Corns ions rWC the silver top of June Grass.
N.Y. (Geneva) Aer. Exp. 51a. lech. Eul. 7. lCOQ.
3;: V?- ~r— #- ‘ . . o I
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C57 ‘f’i-a . - . . -. . . . . .
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Chicago. lCCC.
9Q. \ro
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fungi in syrthetic solutions. am, Jr. Bot.

g9. X: '
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90. ‘9?
"ATEPJIAN, H.J. 1912. hutatfie bij 1331113111ij ..Jmlmm C
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.

actor E. (On. Ast. v. nets-sung pen fe‘slefen
1-"(~. ' ‘ '31 ' -fi I . - . "' rm 1 - 1 -
“atuhlfbhulre, n steruam. 91: 35—53.

91. ‘Y
"U‘E3I3, fiflf. l‘TlO. 2%errirjrtirn‘ of 13‘s :1nfires (n: cerwa L1H fiumgi
in relation to Ph. Ann. Io. Bot. sd. 6:201. lCIG.

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Achromatic Variations in Pathogenic Fungi.

Pllt. I.

LEGENDS FOR

P180 10 30
b.

Fig. 20 ‘0

PLATES I . x11.

Septoria apii on Corn Meal Flask:
‘Eppearance 6? white tufts of
mycelium.

Oladogporium fulzgg on Corn Meal
Fiask. First appearance of the
variant form.

Colletotrichum lindemuthianum Exp.
Eta. Str. II. 531tUre on Corn

 

 

.Meal Flask showing the appearance of

variations.

Colletotrichum lindemuthianug_nxp.
‘ETIT_SEFT—IIT"NOEmaI cfiIture on
Corn Meal.

 

 

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Plato II. A. Cladospgrium‘ggltgg. Parent Strain°

 

A. Mycelium. .
B. Conidiophores and Oonidia.

B. Oladosporium fulvum. variant Form.
0. Mycelium.
D. Chlamydospores.

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Plate II .

 

Plato III. a. Colletgtrichum lingeguthianum. Culture of
the variant'TErm on Corn—Heal.

b. Cladosporium fulvum. Culture of the parent

form on Steafiea Rice.

0. Cladquoriup fulvum. Culture of the variant
form on Corn“fleaIT

   

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Plato IV. Septoria apii. Growth of the fungus on Coons‘
medium varied according to the triangular
system. Complete set of cultures arranged

as per diagram on page 34.

 

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Plate IV...

 

 

 

   

Plato V.
Cilletotrichml lindemuthianun. . Sta. Str. I.
Urowtfi of {He fungus on Coons He um varfed
«owing to the triangular system. Complete set
.of cultures arranged as per diagram: on page 34.

 

Plato V. -

 

 

 

 

 

Plat a VI .

Colletotrichum lindemuthianum;_Exp. Sta. Str. II;
Growth of {He Tungus on Coons MSdium variea In "
composition accaflming to the triangular system.
Complete set of cultures arranges as per diagranm
on page 54.

 

 

 

 

 

 

Plate VI 0

 

 

 

Plate VI I e

Colletotrichum lindemuthianum Exp. Sta. II.

Growtfi o? the fungus on Coons Solution varied

in composition according to the triangular system.
Complete set of cultures as per diagram on page 34,
and descriptions in Table VI.

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Plate VI:-

 

Plato VIII.

Cladosporium fulgum. Parent form grown in light

on Coons medium varied in composition according to
the triangular system.

     

 

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Plate VIII.

 

 

 

PlfltO IX.

Cladosporium fulvum. Parent from grown in the
ar on Coons EeHIum varfied in composition
according to the triangular system.

 

 

 

Plate no -.

 

 

Plat. X.

Cladosarium fulvum. Variant form grown in the
ar on cons e um varied in composition
according to the triangular system. .

Plato X.

 

PlintO XI 0

Cladosporium fulvum. variant form grown in light
on Coons Medium varied in composition according
to the triangular system.

. . point. 1
54. . .

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Plate XI.

 

Plate XII.

Fig. Q. a and b.

0.

Fig. 2. a and b.

Cladosporium fulvum parent form.
under side of the Ieaflets showing
typical infection produced by
artificial inoculation.

Upper side of the leaflet showing
type of spot.

Cladosporium fulvum variant form-
‘Ufider side of IeaTIets showing
type of infection.

 

Upper side of leaflet showing
type of spot produced.

s ll “sir

Plate XII.

 

 

 

 

 

 

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