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ATYPICAL AND PATHOLOGICAL UJLTIPLTCATION O F
CELLS APPROACHED THROUGH STUDIES ON
CROWN GALL1
A. J. RIKER
AND
T. 0. BERGE
(Prom the Department o f Plant Pathology, University of Wisconsin)
TNTROIXJCTION
The basic principles involved in atypical and pathological multiplication of cells seem to need further elucidation if continued progress
is to be made against cancer and related growths. One approach to
this complex and difficult problem is through a study of the activity
of individual cells. Their importance in disease was emphasized in
the middle of the last century shortly after the development of the cell
theory. This study may emphasize tlie physiology (1) of tlie agents
causing atypical cell proliferation where these are known, ( 2 ) of the
proliferating cells, and ( 3 ) the interrelations between the cells and the
stimulating agent.
One of the greatest handicaps in such work has been the lack of
suitable experimental materials and methods. The obvious difficulties
involved in controlled tests on human beings have stimulated the research worker to seek other living materials suitable f o r exacting
experimental examination, but the use of such materials involves the
danger of drawing unwarranted analogies between one kind of living
organism and another. The difficulty of comparisons between cells of
lower animals and those of man is clearly recognized. Obstacles in
comparisons between plant cells and animal cells are likewise evident,
While these difficulties should warn the investigator against unjustified
interpretations, they should not discourage him from employing suitable material and from pursuing lines of investigation which either may
clarify the situation or may indicate the manner of reaching a solution.
Students of cytology recognize that proteinaceons living material is
fnndamentally similar, that studies of plant cells have aided in the
work on animal cells, and r i c e vwsa. It seems so well accepted that
many basic life processes in the cells of plants and animals (irwluding
man) are similar, that this point needs no discussion here. It is likewise unnecessary t o elaborate on the axiomatic expression that a basic
advance in one biological field very commonly assists in the development of related fields.
Certain plant materials offer some distinct advantages over animal
materials to the investigator studying various aspects of the cause of
cell stimulation. Among these the following may be mentioned. (1)
Only a small outlay of time and money is necessary to provide large
1 This ~ u r v e y ,suggested by the International Cancer Research Foundation, was made
possible by grants from the special research fund of the University of Wisronsiii and frolrl
the Foundation.
310
ATYPICAL AND PATHOLOGICAL M U L T I P L I C A T I O N O F C E L L S
311
numbers of experimental individnals. ( 2 ) Adequate statistical studies
of results may easily be made 11rcause of suitable numbers readily
availa1)le. ( 3 ) High genetic similarity of experimental material may
be sccnrccl 11y tlie use either of piire lilies o r of plants obtained by
vegetative propagatioii from oiic indivitllual. (4) Plants a i d certain
plant parts may be grown with com1)arat ive ease in v i f r o under aseptic
conditioiis. (5) Sevei-a1 bactcirial orguiiisms ;IS well as various 11011parasitic factors may stimulate abnormal cell growth i n plants. (6)
Siiiglc-cell isolations of tlicse bacteria asslire the higliest purity of caltures that piwmit methods afford.
Among these cell-stimulat iiig bacteria the best ltnown is the crowngall organism, Phytowionns tzi?nefacieiis (Smith and Town.) Rergey
c.t nl.’ Y l i ~disease which it procluces (Fig. 1) in a great number of
diffcrc>iit plants litis frcqiwii tly 1 ~ ~ 0 1callccl
1
tiitlier a 131a11t tumor or a
FIG.1. CROWN GALL ON YOUNGAFFLE TKEES I X T ~WEEKS
N
AFTER INOCULATION
In such a severe cnse death follows in a short time.
plant cancer. Whether or iiot this is justified depoiids upon dcfinition.
Obviously one may define a tnmor oi. a cancer t o iiicludc o r to t~xclndc
crown gall. Many illogical comparisons liavc been made bet\vccn this
type of plant grox7th a i d cancer i n human beings because of careless
use of the various terms employed. At tlic same time, the combination
of hypcrplastic and hypertrophic growth, mliicli commonly follo~vs100
per ceiit of t he inoculations with the c~owii-gallbact cria on plants, is
bot 11 at~-picaland pathological. If one may judge from past experience
in 1)ioloo-icalstudies, an understanding of tlic fniidamciital came of this
”.
stimulat ion in p l m t cells should prove useful t o investigators of cell
stimulation in animals and miiii.
The physiological cause of cell stimulat ioii in crown gall is doubtless
some factor o r combination of factors resulting from the metabolism
of the causul bacteria within the host. Crowii-gall bacteria enter
through aii injury aiid in some niicktermiiied way dist nrb tlie normal
mctnholism of the cells. Several other bacteria (discussed later) in? Otlicr nniiirs conmionly applicd to this organism a r e : Bacterium tu?ncfac9ielzs Smith and
‘IOWIJ.
and ~ S ~ l L t ~ O J l l O t l tLu
f St i i c / w m ~ . s (Smith aud ‘ Y O ~ V I I . )Puggar.
312
A. J. RIIZER AND T. 0. RERGE
duce similar cell stimnlation in plants. Various abnormal growths may
also be caused in plants by a variety of noii-parasitic factors, including
mechanical injury, distnrlied water relations, and interrupted traiislocation of food materials. Altliougli a parasite is associated with cell
st imulatioii in crown gall and provides a very coriveiiient biological tool
t o assist the iiivcstigutor, the fact of parasitism shonld not obscure the
search for the physiological c;~iis(~
respoiisiblc for the atypical ancl
1)athological cell multiplicatioii, t ~ the
s
basic priiiciplcs underlying this
excessive multi1)licatioii of pliiiit c+cllsdoubtless have much in common
with tliose concerned in ahnormal aiid pathological proliferation of
animiil or human cells.
I3ecause of the experimental advantages offered by plant materials
and because of tlic probability that a fuiidamental coiitribntioii eluciclatiiig tlie cause of cell stimulation in o m field may clarify tlie general
1)roblem mid perli~1)sassiet with its solutioii in another field, it lias
appcarccl desirable in the prosent puper (1) to review critically the
pertinent work doiic with crown gall a i d related diseases and ( 2 ) to
consider the possibilities of cwiitiiiucd rcseawh in this direct ion.
ANATOMY
The morphological relations of crown-gall bacteria and the host
{issue will be given brief considcration before taking np the physiological aspects of this aiid related diseases. No attempt is made to
c41borate 011 thc comparisons of anatomical features between crown
gall a i d tlic various types of animal tumor or cancer. This subject
was Ircatcd in great cletuil by Erwin F. Smith (1920b and 1922b), who
reviewed many papers on tlic subject, and lias received more or less
critical coiisideratioii hy n number of otlicr workers iricluding Aulcr
(Kl24), Hcnsaiide (l92S), Butler (1930), Centaiiiii (1929), Hamdi
(I930), Lcviiic (1925u, 1930, 1931), RIagrou (1%8), Robinson (1927),
;113d Stapp ( 1 9 2 7 ) . ~
Some writers have stated that tlierc is rlose histological similarity
bctwecn tissue growth in crown gall and that in certain tumors o r
cancers in animals including miin. Others have thought that snch
compaiisons were too far-fetched to be worth considering. As alrcady
meiitioncd, mnch of the discnssion seems to have developed over diffcwiiccx in definitions. It is not the purpose of the present paper t o
(lobate whether crown gall is or is not a tumor or a eaiicer. The senior
writer has avoided such comparisons while rcyorting work on crown
gall and related dixcascs both hecause of tlic danger of unwarranted
iiiialogics arisiii(>*
from varyiiig definitions, ancl hecausc of the probak
bility of obscuring by such discnssioiis the basic problem co~icernecl,
i . ~ .tlic
, physiological cause of tlie atypical aiid pathological multiplication of cells. In the present treatment of anatomical features milch
is omitted wliicli liiis been fully covered in the reviews mentioned above,
a d cmphasis has becii plncctl npon some of the more recent develop3 Wlicn two or morc citations arc made, thcir scqucncc is arrangcd alpliabetirally secording t o author and has nothing to do with tho merits of the papcrs.
A T Y P I C A L A N D P A T H O L O G I C A L M U L T I P L I C A T I O N O F CELLS
313
mcnts and upon those which seem important in relation to the physiological considerations.
Esdrancc and Location of the Bacteria: The path of ciitrnnce of
c~o\v11-gallorganisms iiito the host in sucli a way that iiifectioii is illduced appears t o be tlii*ough wounds of o m type or anotlicr ( Kaiifield,
1928 ; Miincie, 1926; Patty, 1930; Riker, 1 9 2 3 ;~Robinson and Walkden,
1923 ; Smith, 1916e ; and Stapp, 1933). Several workers iiicluding
Riker, and Robinson aiid Walkden, have found that bacteria traveled
some distalice in the tracheae, but did not induce cell stimulation uiilcss
released into the parenchymatous tissue. P a t t y reported that bacteria
uw*eable to enter iiito the stomata of sucli plants as tobacco, geranium,
and castor bean, but werc uiiablc t o inclixce infection in the absence of
an injury. The necessity of ~vonntledtissue is important in consiclering the interaction of parasite aiicl host cells.
The location of bacteria i n the host tissue after having been introduced through injuries has received considerable attciition. Riker
(1923n, 0 ) aiid Robinson and Walkdeii (1923), working iiidcpendcntly,
reported that the bacteria were located primarily i n tlie intercellular
spaces and vessels. IZilrer ciiltivated the bacteria from their natural
position in tlie tissue by mounting fresh sections in agar, and observed
the appearance of colonies. The h c t ex-ia were identificld hy inoculatioiis iiito plants. Pinoy (1925) raised the question whether Rilter had
not obscrved Bacillus ~ / I I ~ T P S C P in
~ Z the
S
intercellular spaces instead of
crown-gall bacteria. Robiiisoii and Walkdeii found large numbers of
the organism also on the surface of the gall. Various parts of the
work by Rilrcr and by Robinson and Walkden on the position of the
bacteria liavc beeii repeated aiid confirmed by several investigators
including Haiifield (1934), Rerridge (1930), Hill (l928), Rlagrou (1926),
mid Sylwester and Countryman (1933). A iiumher of these writers
found the bacteria more abundant on tlie surface than within tlic galls.
The bacteria have also been found inside large restiiig exterior cells by
I'inoy (1925) examining blagrou's preparations, by Magrou (1926),
and others. Ho\?T~vcI',
confirmatory evidence is laclring (Smith, 1920a,
and others) that the hacteria o c c i ~ rwitliiii the small rapidly dividing
cells, its reported by Smith (1912, 1916a, 1916c). ("ertain writers consider that tlie bacteria 011 the surface of the gall, others that those within
the tissue, a r e primarily responsible for cell stirnulation. It was fouiicl
that masses of bacteria placed on the surface of susceptible stems iiiduccd no proliferation (Rilrer, 1 9 2 3 ~;) aiid, furthermore, bacteria
placed upoil the snrfacc of actively proliferating callus tissue (Riker
and Raiifielcl, 1932; Xiker and Keitt, 1926) commonly had no added
effect. Otlici- trials on the siirface of pith cells in castor bean stems
and petioles (Rilrer, 1 9 2 3 ~ )showed no proliferation except where the
tissue was injured. From tlicse results it appears more lilrcly that tlic
bacteria within the tissue, i-atlier than those on the surface, a r e responsible for the stimnlatioii of cell proliferation.
The movemeiit of the bacteria within the host tissue, after entrance,
has been studied from several standpoints. The bacteria were rek
?
314
A. J. RII<ER AND T. 0. UEILGE
ported 1'4' Rilicr (1'323~)to t r a ~ t~l f c w cciitimetcrs within tomato
tissue wlieii a contiiiuoiis cliniiiiel of liquid w i s l)rovidcd. H e coiisidcrccl that tlic motility of the bactclritz might he ail impoi*tarit factor.
Im4iie (192571) f a i l d , in limited trials, to secure sirnilnr migration i i i
rnspberi.y. Hill (1928) st at cd that bacterial zooglocae grew during
several lioui~sfor ii fern millimet crs tlirougli the iiit crccllnlar spacer.
'I'liclse studies liavc bccii estcnclcd by Hill, Rrittiiigliam, Cribboiis, tirid
Watts (1930). Howcver, Ivaiioff' aiid Rikcr (1930) f ouiid tliat pliciiiomeiia similar iii apl)(wraiicc occnrrcd in a few minutes wi tli dwtl
bacteria mid with pirticlcs of India ink. C:onscqucntlp, they coucluded
that various fact oix, iiicliicliiig negative pressure and calillarity, alionld
be coiisidclred. For movcmciit iii older galls, Smith (1932b, p. 44) lias
given perlial~stlic best s n o v s t ioii, following his descript ioii of a. small
h?
pith tumor not (wiiiic( 1 with the primary tumor. While adliering l o
tlic idea of cell stimu
on by iiit raccllular hact cria, lie speculat ccl 011
iiitcrcchllnlar movemciil as follows : "TVc may suppose t h e small pith
tumors, if tlicy a r e i*eallysecoiidary, origiiiateil in this way ; that during
tho t eariiig opcw of n.ootl mid pith, resulting from the rapid growtli of
1l i c i 1)rimary tiimor, cvrt ain of its iiifcctcd cells wcrc~(wished liberutiiig
i i i t o the womiclcd wct ; i r ~ asomc
,
of tlic motile rods of tlic parasite wliicli
i l i c ~ i imade tliciii. way tlirougli iiitcmellul;xi* s1)wcs or fissures into a few
of llic to]-11 pith cdls aloiig tlie Iiiic of tlic rupture, converting these
c ~ ~ liiito
l s a tlozcii 01' more iicw centers of tumor growtli." Tlic movcmcnt ant1 location of ihc bacteria ('a11 also bc htiiilied by the cff'ect on
the various iissiws stimulated.
Cell Growth aOou2 t h c Bacteria: Tlic types of tissue stimulated by
crown-gall bact ci*ia and the diuract e r of growtlis resulting from such
stimulation liuvc I)eeii studied in many plants by a iiumber of writers,
(1925a,
including Butler (1!)30), Clooli (1923), Hamdi (1930), I~c~viiic
b ) , AIagrou (1924), Iiiltcr (1937), and Smith (~iiimcroiispaj)crs, cspccially 191&, 1922b, niicl 1928, wliicIli clitlicr review o r rcfcr t o earlier
work). Smit li (1928) concluded Ihat many of the commoii t crat ological
forms in plaii t s a i d animals develop becansc of p r a s i t i c o r mcclitmical
displacements occuniiig in early life. Fiirthcr considerat ioiis of tlict
various featuws iiivolvcd arc omitted bccausc of space limitation. It
is geiiei*ally coiisiclcred that aiiy living plaiit tissue of a susceptible
host may hc stimulated t o growth. While the more active tissues like
cwnbiurn and phellogcn commonly react more readily aiicl vigoroiisly
than more cwmplctely cliffcrciitiated tissues, aiiy living group of cells
by reiicwccl activity may indicate tlic positioii of the bacteiia.
Tlie rclntioii 1)ctn~rerithe positioii of tlic ha rj;, aiid new giwwtli is
quite c3leai. iii tlic. cbarly stages of gall foiamatioii. Bilicr ( 1 9 2 3 ) ~ i i c l
Itobiiisoii aiid Wullidc>ii (1923) showccl tliat rapid divisioii occnrrod in
the cells immccliutc1ly sni.romiding tlic buctcrin. TF711c11 thc c+ellsbcgaii
1o divide, the eai*ly divisioiis werc1 fi*equciitly iiiicqual. Tlic daughter
cclls w ~ r cso oriciit eel that the i i c v walls were commonly laid clowii
cwmpi.atively near tlic position of tlic bacteria in a plane faciiig i t ,
as sliowii iia Fig. 3. Compnrablc obscrvatioiis htivc becii reported by
ATYPICAL AND PATHOLOGICAL MULTIPLICATION O F CELLS
315
other writers including Rerridge (193O), H a m & (1930), Leviiic (1925b),
and Magrou (1927). Altliough statements hare bceii made (Centanni, 1929 ; Jciiseii, 1918 ; I<auffmaiiii, l927b) tliab thc~bacteria disappear from the older galls, Aluncic (1926) and Rikcr aiid Keitt (1926)
isolated bacteria consistently from suit able samples of old crowii-gall
tissue. Tli(1.y reported, however, that it was possible to secure small
fragments of crown-gall tissue which coiitaiiicd 110 viable h c t e r i a . It
appeared tliat the bacteria occurrcd iii small pockets from which they
could he released by macei*ation. The entire exterior of tlic gall could
be safely surface sterilized or cut away. However, negative results
were obtained when the small iiitcirior portioiis used for isol nt'ions mere
FIG.
?.
INTRKUELLULAE SPACE ("ON1'AINI"G
CltOT\TN-GAI,L B.\CTEXIA,
EI(;HT DAYS AFTER
1 NOCT1I.ATIOh
Individual rods a r e not distinguishable in the dark inass, a. Tlic largr tomato pith cells
havo laid down new ivallu a t b. Tho nuclei, c, liave apparently been attracted toward the position of the bacteria. The essciicc of the problem is t o detcrminr, if possible, the physiological
cause f o r the hcgimiiiig of cell proliferation, :rnd for its rontiiiuatioii. (After Riker, 1923h.)
treated either with a flame or elicmicals, especially tliose coiitairiiiig
mercury, because t lie bacteria were thus killed. In illis coiiiicclioii, it
should be observed thal various iion-pai*asitie fact 01's may iiiduce overgrowths similar i n appeai-ance to erowii gall. Also, bad cria may iiiduee an effect similar to girdling and result in wouiid overgrowth tissue
containing n o cell-stimulating bacteria (Rilier aiid Hildebraiid, 1934).
The pertiiicnt qnestion of ai~toiiomous growth is discussed under
"Physiology of thc Host" aiid is omitted from these histological coiisiderations.
The histological examination of older crown !all shows a disorcwiized
mass of hyperplastic and hypertrophic tissues more o r less
h
iiitcmpersed with badly or.ganized groups of vascular elcmciits 1.e-
316
A . J. ILIREIl. A N D T. 0 . BEliGE
scmbliiig t rnclicac (Fig. 3 ) . Tlio size ~ i i dshape of tlie small, rapidly
dividing wlls aiid lliohc maltiiig u p the liypcrplastic aiid hypertrophic
tissiies vtiry somcwliat with llic age of the gdl, tlie kind of plant, and
tlie coiitlitioiis uiitlci* wlii(a1i the plant was growii. In maiiy cas‘c’s il
appears (cb.g., Kiltci., 1!)830 ; Smilli, Krowii, aiitl Rlc(”ulloc~11,1912)
1h i t the 1iyperl)lastic tissncs liuvc clcvcJ1ol)cclfrom suhdivision of larger
cells. This is ospccially clear in tlic earlier stages of c*rowiigall where
ILiltcr (19230) lias studied 14ic division of cells bordering the posilioii
of tlic bacteria i i i tlie iiitcwellnlar spaces. Tlic histology of crown gall
h i s bccw so \veil covcrcd by various worlters, inc~ludiiigHam& (1930) ,
Liviiie (19%5n, 1930), Jlagi*on (1927) , Riltcr (1 933a, 7, ), IioBiiison and
Wnlltcleii ( l 9 2 3 ) , Smith (1912 t o l 9 2 2 ) , and Sylwester and Couiitrymaii
(1933), that it is omillcd lic~rcin f a w r of a more complete discussion o i
rcporls on “metastasis.”
“,So(~owlar,y1 1 ~ 0 ~ / o ~ . ~arid
s ’ ’ “ T ~ [ w oSfrarids”:
v
‘ Secmidt-\rytumors ”
may be induced with cvxq)arativc c~ascin a vai.icty of cliffcrciit plant s
11y iiioculal ions 011 rapidly elongating tissues. It lias been statccl
(Smith, Browii, and AJcTullocli, 1912, p. 15) tliat, “soon after the appearance of a primary tumor, particularly if tlie pluiit [Paris daisy]
is will iiourislicd and growing rapidly, tumoi. strands pnsli out of it
iiito tlie normal tissues, gclici-ally, it would sclcm, tiloiig lines of least
iwistaiicc. ” T h u s socoiiclary tumors wei+c formed at various plirccs.
J’c4tlo (1915), working with siiiiflowc~,sccurecl similar rcsulls. Ku
(1913) a i d Nemcc (1913) ciuestioiicd this iiitcryrctntioii of tlie origin
of ‘ secondary t ~ n l o r ~ .A~ ’tlif.f‘ci.ciitcxplaiiatioii of the formatioil of
llicse developments has iiidr~~ciidciilly
liecii provided by Biker (1923b)
iiiid Robiiisoii aiid Walkcleii (1983). ‘I’licy fouiicl that ‘‘ secondary 111mom ” occurrcd rcizdily w2ieii siisccptiblc plants willi apical buds (wit aiiiiiig a iiumber of closely coiideiiscd iiodcs aiid iiitcriiodcs ( I ’ w i b
daisy, suiiflow~~r,
s~veelp a , aiicl lol~cc’o)were iiiocwlatccl at tlic to1)
of the sl em. These rcsulls were iiot sccurcd v7lic1ii snscqliblc pltnits
such as tomato, wilhout coiidciised apical buds, w‘cm tlius inoculat
As the iiccdlc l)uiiclure was made iiito the top of a stem, liquid from
rupturc.d cells moved for a short distance through ilic iiitercclluliir
sl~accs. If 1)artoria U’CTC‘ iiitrocliicccl with tlic iiiocnltrtiiig iiccdlc, tlicb
niovcmeiit of tliis liquid cloubtlcss servcd t o distri1)ute the bacteria, tis
cliscnssed earlier. Iii any (*ase it 1)rovidccl a liquid c~litliiiiclfor 1 1 1 ~
migratioii of tlic ci-own-gall I w ~ t c r i a . Altliongli tlic distttiicac of spwtitl
of the released liquid was very s1ioi-t aiicl in extreme ~ a s e s;imounlcd t o
oiily a few millimeters, it wizs sufficient iii llicb cwiideiised buds t o c’ar~y
*iainto a iiumbc~rof iioclcs. Sometimes with SC‘VCIT iiiocdu1ioii the suhscquciit grotvlh of crowii-g;fill tissuc piweiitetl or reducc~l
tlic eluiigtitioii of tlic iiitcrnodcs (Riltcr, 198X1). In otlic~’cases, t l i c
1issue iiivaclccl by llicl b a d criu w i s elongated by the dcvclopmciit of
the apical bud aiitl rapid lciigthciiiiig of tlicl internodes. la this miiiiiic‘r
tlic bacteria ~vvci*c~
distributed for some distance aloiig tlic stem. IYI
pltuit s like. siiiiflowcr, Iiilt~r(19231) foniid secoiidary galls as far as
13 iiitcmioclcs aiicl R clistaiire of 49 (am. from the.y)oint of cntixacc of the
ATYPICAL A N D PATHOLOGICAL M U L T I P L I C A T I O N O F C E L L S
317
bacteria. ‘ I f4ecoridai.y galls ” developed after a suitable incubation
period at points presumably recriviiig a sufficiently large iinmber of
bacteria to induce i*elativclp rapid proliferation. Tlie esseiitial features of t his interpretatioii of tlie mecliaiiism by wliicli “ secoiidary
tumors ” ap1)c’ar liavc 1)eeii confirmed (Hamdi, 1‘330; L e v h e , 1925b ;
aiid Stapp, 1927). Similar results w c w obtained by hfagrou (1928,
1). 560), \vlio eonsitlei.ct1 this explanation possible in certaiii cases, but
who said (trxiisltii ioii), “. . . this method of inociilatioii appears somewlint diflciwit from that by i~liieliSmith obtained metastases i n Paris
daisy ; it swms, awordiiig t o t lici &wription of his esperimeiits [Smith,
Bi*owir, aiicl Alc~’nllocli,19121, tliat lie iiiocnlatcd the sttlm in a youiig
wxioii, but at some distance from tlrc tci*minalmerist em.” This statc-
ii~c
lip Smith, I3row11, aiid ,11c(‘ullocli docs
ment of tlic p i ~ o c ~ ~ d icmploycd
iiot follow tlicir ow11 a c c o i u i l (1912, 1). 23) : “At the time of the inoculation t lie stems T V C ~ Y soft a i d rapidly elongating, and tlic needle pricks
were made in what wits tlieii ilie t o p of the plaiit.” Several writers, incliidiiig Riker (1!)236), Robiiisoii aiid ‘Clialkdcii (l923), aiid Stapp
(1927), have failed t o eonfirm tlie report (Smith, Browii, and Alc(:ullocli,
1912) that the seconclary tumors necessarily repeated the structure of
the primai-y tumor a n d tliat there was always it connecting strand.
“Tumor straiids,” according to the above interpretation, were
formed by the cli\4sioii of cells aloiig cvi*taiiiof t l i ~clongatcd cliariiiels
of bacterial movement. I n tlie process of elongation of the internode,
thci number of bacteria was doubtless considei~ablyreduced in certain
portions. As a result, witliiii tlie given time of incubation, the amount
318
A. J. RIIZELZ A N D T. 0. BERGE
of proliferation was relatively small, especially in slowly reacting tissue.
Such development gavc tlic appearance of “tumor strands.” I n some
cases it w a s cxcecclingly difficult t o fiiid a strand connecting the point
of entry wit 11 the “ sccondwy tumor, ” but Smith, Rrom711, and RIcCulloch
(1913) rcported “tumor strmcls” in every case examined. Lcvine
(192511) cxumincd eases in wliicli lie was niiablc to locate a connection.
I t a p p i r s from tlic evidence available that Smith and his associates
(l!H2) gave a dcfcctivc iiitcrprctation to the origin of “tumor strands.”
Magron (1924) considered that tlic tumor grew by seiiclirig from its
margin, iiit o the sii t*roiindiiig parmicliymat ous t i ssiie, out growths
formed by dceply staining small cclls. Later, Smith (1922b) modified
somcwliat this concept of invasion to anotliclr of growth by successive
transfer of the growth stimnlus f m m tlic tumor cell t o its neighbor.
Rucli tLppositioiia1 growth in (*row11gall was discussed at length by
Smith (I922b). He showed that the c*hangcfrom typical cells to tumor
cells had occurred in sitzc by tlie conv(mion of swollen but otlicrwisc
normal cells in to congc~icsof small tumor-like cells, visibly siirroundccl
i n many cases by tlic strctclieci wall of tlie oriFina1 cell. He considcrctl
t liat this was tlic u s n ~ 1manner of giwwth within iiifccted tissue, and
that iiivasioii of ncw tissuc occui*r.cclby appositioiinl growth, in one
t l i i ~ ion
d only (end-to-clncl). l’lic division of larger cells into smallcr
oiies uiic1c.r the influcwac of the b a deria lias also been observed by
including IIwmdi (1930), Iifagrou (1928), and Biker
s w c ~ - awriters
l
(19230). Ccrtain cells resisted division aiid remained as giant cells
with interesting cytological cliaracters in tlie midst of hyperplastic
1i s su c.
Cj-/tologicnZ Studics .- Abnormal nilclear pliciiomcna liavc bcen rcl)oi*tcd iii various studies of crown gall by Smith aiid his associates
(Smith, 191Gn, c, d, r ; 19200; 1923a, 0 ; and Smith, Brown, and hlc( ‘iillocli, 1912 ) . RI ost of I lie work by other iiivestigutors, inchcling
H;ii*slil)crger (1917), Lcvinc (1!)2r>a,1930), hl agrou (1%4), and R i k c r
( 1927), serves t o corroborate aiid estcwd their findings. Nuclear iiiim1 ) c w of 3, 3, 4, 8, and even 10 to 30, liave hcc~iireported in a single
c*rowi-fi’ullcell. Tlicl l:i~*gclriiirmhcrs were found less froquently. A4nlt inuclentc cells Ilavc also occasionally 1)een found in adjacent normal
t issne. Lobed nuclei snggcsting amit ot ic division liavc somctimcs bccln
o1)scrvecl. A progiwsive reduction in tlic iiucleocytoplasmic ratio w;is
olmwocl in giills on tom;ito, tipproaching ti minimum near tlic tliirtyt liircl clay after inoculation, from whieli there was a slight return.
ri
J lie caliromosomc number iii ci’ow~igalls of 1)ec:ts has been counted
by Lcviiic (1925n, 1!)30), wlio foniid that mdtiples of the haploid number ( 9 ) occul*rcd in varions cells. Tlie increased chromosome number
of‘ c ~ l l sin the affcctcd area, with increased genes f o r growth, was
tliouglit by Wingo (1928) t o promote ex(vxsive cell development. Thus,
1 i statrtl
~
(1). 41X; t~mislatioii): “ If we iismme purely scliematically
that a cci*tainHf’ta plant cwntains 4 genes specific f o r growth promotion
;iiid 1 inhibiting gene, Ilicn the Ictraploid cells will posscss 8 t o 2, or
(imow gi.0w tli-1)I’()moti 11g genes t1i;rii grow tli-inhihi ti rig, while the nor-
ATYPICAL AND PATHOLOGICAL MULTIPLICATION O F CELLS
319
ma1 type only has 3 more growtli-promoting genes. On this basis the
increased growth-energy of tet rnploid cclls may be explained.” He
saw as not iiicoiisistcnt wit11 this theory the fact that tctraploid cells
occiirred occmionally in some types of plants ancl regularly in others
without iiiducing tnmor f’oiemation, since tlic kind of tissne as ~ w l al s
the biotypc of tlie orgaiiism i n which tlie abiiormnl ccll arose cxcrtcd an
inflnence 011 -\dietIiei. and in what miiniic~these cclls wtbre able to clcvelop. I’olyploidy \WR o h s c ~ r c c lby Kost off aiid Kendall (1933) in
crown galls, in ovc~~gi*owtIis
caused l)y chemicals, and i n sl)oiit:ineous
tumors 0 1 1 Iiybrid to1)awo. They consider tliat polyploicly vaiiiiot be
treated as a cause f o r tumor gi.owtli, bnt may bc a harmlc
of conditions iiiitiatctl iii tumoiwus rcgioiis. TjVhitalicr (1934) rcported
a correlation in some cases of’ ;L cliaiigc in c1iromosomcl number wi tli
the occiirrcii(~eof iioii-l)nrasitic t umors on grafted t ohacco. It is clifficult to acccpt the suggcstioii tliat mnltiiiuclcutioii may be rcsponsil~le
f o r the growth of crow11 gall. It c.lc~ai.lyis iiot responsible in the initial
stages illustrated i ti Fig. 2. H o w c v ~ r ,t Iic w.ell known control which
chromosomcs csc1.t o v ~ gi*o\vth
r
suggclsts Iliat tlir (aliromosomes may lie
inflnenccd by the causal :igcnt.
Various types of ccll inclnsions liarcl bceii observcd in crown-gall
tissues ; c.<q.,IZiliei* ( I 927) ol)semed various crystals, some of which
~ w r eprobably calcium oxtilittc, and several clifferent undetermined
amorphous bodies. Rome of these RIiloviclov (1930) considered were
probably tannin.
Altliongh statement s ocwsioiially a p l ~ a rs i i g p t i i i g that certaiii
of tlic mor~)liologicalancl cytological changes found in crown-gall tissues
may liaw causal ~ o n i i ~ ~ ~ f iito nseems
s,
morc liliely that they arc tlic
resnlts of ])at liological conditions follo~vingphysiological disturbances.
It is important t o uiidcmtund them as clcurly as possible, hccansc thesc
effects ma>- suggest the factors by wliicli tlicy are induccd. With this
in miiid, vurions 1)lipsiological chaitgcls which h a r e hecii rcyorted a s
induced by crown-gall h i e 4 cria or a s occiii*ringin carom-gall tissne arc
considered in some detail.
P~IYSIOLOGY
I’li yx iol ogy of (’t.ow,z-gall Ba(?P r i a
r i
1 ho p1i;vsiology of the cro\vn-gall bacterium has ~ ~ c c c i ~mucali
- c d attcntioii. 111 tlie olclcr litcbrature some contradictions a r e foimd in the
clcscri~~tions
by varions writers of tlic cliaracteiktics of this organism,
due, iii 1)twt, no doubt, (1) t o confusion between Pl/ytonio?zn,s tumr.l a c i tt s and Rac il I I s radio 0 a( Y Heij. and Van Ileld., o r the morc
recontly dc~scribed1’. d i i m g w f ~ sR. R. 11’. I<. and S., or some unidentified organism, and ( 2 ) to 1)ossible variations i n different crown-gall
strains. Smith, Brown, and To~viiscnd (1911 ) o1)scrvc.d sti+ikiiig diffcreitccs bet ween different isolations of crown-gall bacteria. Hcndrickson, Raldwin, aud Rilwr (1934) have iioted variations in single-cell
cultures. It h a s already h c n lmiiited out (Rikcr, Ranfield, Wright,
320
A. J. RlKRR A N D T. 0. BEltGE
K c i t t, aiid Sageii, 1980 ; Wright, Hc~iidrickson,aiid Biker, 1930) thtd the
poured plate mctliocl did riot prove reliable f o r the separation of the
crown-gall organism from other associated bacteria. C)onsequently, in
tlie following consideration of t Iic. physiological propcrt i cs of this organism, attention is given t o the work wliicli lias been 1)asecliipoii single~ results of sucli work arc available. An
cell cultures, ~ v I i e n c wtlic
niiderstanding of the physiology of cc.11-stimulating organisms, wliieli
may be rcsponsil)lc through their mctabolism f o r tlic abiioi*mal growth
of plant tismc, scclms iml)ort:int if onc j s studying the basic cause for
atypical and pathological multiplicaation of cells. Tlic so~irccsof carbon
available fo tlici bacteria arc considered first.
U i m ~ i c a lSubstawcs Usctl l q ('t~ll-sfi~?l;ulatiwg
Buctcvia: Tlie ntilizat ion of car.bohydi~atesaiid rclatctl substances as a s o i ~ i -of
( ~carbon,
a s slioww by qimiiti t ntive dctcrmiiiatioii of sugars in tlic mcdium tuicl
1)ycliwnges botli iii liyrlrogcii-ion c~oiicciitrationand in titrat able acidity,
wis stuclicd by 12ikcr, Haiificald, Wright, Kcitt, and Sagc.11 (1930) i n a
liquid peptone-salt mcdium coiit tiiiiiiig. 5 g. of tlic soiirce of carbon per
lit cr, incnhated at 21" C. f o r tweiity-oiic days. The pcrcciitagc of sugar
fwmeiit ccl by crowii-gall bacteria iii sucli a medium ~ 7 a :s arabiiiose GG,
xylosc 52, gluc~osc~
32, g:.alactosc 43,maiiiiosc 66, fructose 34, sii(*rosc51,
multose 35, aiid lactose 15 p r cent. No correlatioii was found between
tlic amount of carboliyclrat c f e ~ m c n t e dand the cliarigcs in citlier hydi*ogcn-ioncoiiccntrnt ioii or titrat:.able acidity incluced. With nrabinose,
sylose, rhamiiosc, galactose, maiiiiose, fructose, sucrose, maltose, lact ose, raffiiioso, mc.lczitosc, szilicin, cwuliii or maiinitol, little or no cliiiiigc
i i i 1)H took place. A slight iiicrcasc in acid reaction occurred with
glucose aiicl clnlcitol, while a decrease resulted with starch, destrin,
iiiuliii, or or.ytIiiito1 a s R soiircc of cwboii. As measured by total
titratable acidity, 110 sigiiificant c*liaiige was iiiduccd with ;mil)inose,
xylosc, rhamiiostb, glucose, g:ilact osc, maniios~,mcrosc, maltose, laclow, ciextrin, saliciii, dnlcitol, or mtiiinitol. A slight titratablc acidity
;~ppcarcdwith fructose ; aiid a titratnhlc alkalinity occurred with r a f fiiiose, melczitosc, sla1~11,iiiuliii, cscdiii, o r erytliritol. Comp;ira?)lc
i-csults wcrc sccurucl by 3luiicie aiitl Suit (1930) and Suit (1933). The
;ilkaliiiity iii those (*;isc's
proha1)ly tlic rcsult of ammonia formation
from pcptonc.
Tlic fcrmcntntion of various S O U ~ C ~ofS carbon w a s further studied
i n a miiic~r*til-saltmedium coiitaiiiing potassium nitrate as the source of
iiil rogrn. This proecdurc avoided the complications resulting from thc
iitilizatioii of tlic c~urboiiin tlicb pcptonci. Tlic cultures were iiicuhatc4
foi. twenty-oiic days at 28" ('. Good growth was observcd ( S a g c n ,
13k r, iiii ci HHI tlw i 11, 1934) wit 11 tmibi ii os c, glnco e , lac t o sc, s11cro sc,
r'itffiiios~~,
clcxtriii, saliciii, ethyl alcohol, prop$ alcohol, glycerol, maniiitol, dnlcitol, laclositol, ai+abitol,sodium acetate, sodium propionatc,
lnctic acid, calcium glucoiiatc, sodium sueciiiatc, malic acid, o r sodium
cit rate. Slight growth oc*currocl with adoiiitc, butyric acid, glycollic
acid, stcaric acid, sodium maloiitite, o r ttirtaric acid ( a s sources of
cnrbori) ; a trace of growth with melliyl alcohol, bntyl alcohol, quercit
~
ch
$
7
2
~
A T Y P I C A L A N D PATHOLOGICAL M U L T I P L I C A T I O N O F C E L L S
321
or sodium formate; and no growth with sodium oxalate or with no
source of carbon in the substrate. While little or no acid reaction
was induced in the utilization of carbohydrates, a n acid reaction resulted
with ethyl aiid propyl alcohols, and a basic reaction with the salts of
organic acids. Similar studies were made on nitrogenous compounds.
The utilization of various sourees of nitrogen was studied (Sagen,
Riker, and Baldwin, 1934) in a similar mineral-salts basal medium.
Glucose was the source of carbon, and various nitrogenous compounds
were added. The cultures were incubated for three weeks at 28" C.
Increased turbidity and changes i n both hydrogen-ion concentration
aiid titratable acidity were noted. When inorganic compounds were
utilized as sources of nitrogen, good growth was obtained with ammonium sulfate, ammonium citrate, ferric ammonium citrate, ammonium nitrate or potassium nitrate ; a trace of growth with potassium
nitrite ; aiid no growth with potassium nitrate in the absence of a source
of carbon. Upon the addition of organic nitrogenous compounds, good
growth was obtained with methylamine, acetamide, oxamide, succinimide, dicyandiamide, asparagin, urea, uric acid, glycine, dl-alanine,
1-tyrosine, 1-cystine, 1-aspartic acid, d-glutamic acid, or 1-leuciiie. Little
change in reaction was noted after utilization of most of the nitrogenous
compounds. With ammonium sulfate, potassium nitrite, oxamide, 1tyrosine, 1-cystine or 1-leucine, however, a n increase in acid reaction
was found. Distinctly alkaline reactions appeared with ammonium
citrate, succinimide, urea, dl-alanine, 1-aspartic acid, d-glutamic acid,
peptone, or potato extract.
No nitrate reduction by the crown-gall organism was reported by
Muncie and Suit (1930) after incubation for two weeks in nitrate broth.
Sagen, Riker, and Baldwin (1934) noted a slight reduction of nitrate to
nitrite where glucose, dextrin, ethyl alcohol, glycerol, mannitol, adonite,
sodium propionate, lactic acid, calcium gluconate, sodium succinate, or
malic acid, respectively, were used as the sources of carbon. No material difference was rioted in nitrate reduction by cultures kept at 25"
and 32" C. respectively. These temperatures lie on either side of the
critical range (28" to 30" C.) for pathogenicity on tomato. I t appears
that nitrate is not reduced by this organism much, if any, beyond the
point where it is utilized for bacterial protoplasm.
Fixation of atmospheric nitrogen by the crown-gall organism was
reported by Israilsky (1929), ~ 7 h ogrew the organism in a medium containing 20 g. of manriite and 0.2 g. of dipotassium phosphate per
liter of water. Schroder (1932) observed a small increase in nitrogen
in Pkyfomoizas tumefacirns after thirty days in a liquid medium containing glucose arid mineral salts. The amount of nitrogen-fixation
was so slight, however, that she attributed it in part, a t least, to absorption by the medium of atmospheric nitrogen through the cotton plugs
used. Sagen, Riker, and Baldwin (1934) in their first experiments
found crown-gall cultures able to grow on agar media without the
addition of any known nitrogen. However, when the agar was washed,
no growth was secured. This showed that nitrogen had been available
322
A. J. RIKER AND T. 0. BEROE
in the agar, and that the bacteria wcrc unablc t o utilize atmospheric
nitrogen. I n prcliminary trials Pinckard (1935) secured growth in
liquid media to which no nitroFen was irit entionally added, but when
the water supply was doubly distilled no growth was secured.
These studies on nutrients available to the crown-gall organism
become more significant when a comparison is made (1)with thc nutrients available in thc host plant (considered later), and ( 2 ) with
parallel trials on other cell-stimnlatiii,rr bacteria and on closely related
non-pathogenic organisms. While it is unsafc t o assume that evcry
bacterium in this group necessarily stimulates abnormal tissue growth
in the same way, still it seems that further studies on the characters in
which they arc similar appear more promising than studies on thc
characters in which they arc unlike. Corrcspondingly, studies on the
characters in which non-pathogenic cultures are diff ercnt from the
pathogenic cultures appear more promising than work on the characters in which they are similar.
Comparisons of the physiology of certain bact eria in this ccll-stimnlating group bavc been made by Sagen, Riker, and Raldwiri (1934),
and by Hendrickson, Baldwin, and Riker (1934). In both investigations
Phytornonas rhixogenes R. B. W. I<. and S. and Bacillus radiobacter
Beij. and Van Dcld. werc carried in culturcs parallel t o P. tumefaciens.
Much more cxtensivc comparisons were made by Pinckard (1935), who
employed: P. tumefaciens; P. beticola (S. B. and T.) Bergey et al.; B .
savastanoi (E. F. S.) Bergey et al.; P. savasta~zoivar. nerii C. 0.
Smith ; and an unnamed raspberry cane-gall organism. These comparisons involved a number of characters, including pathogenicity,
utilization of various sources of carbon and nitrogen, tcmperaturc
relations, dye absorption, variability of the bacteria both in cultures
and in plants, limiting rcactions for growth, and changes induced both
in pH and Eh. A detailed comparative consideration of these characters is omitted bccausc of space limitation. Some of the determinations, discussed latcr, have definite beariiig npon the validity of certain
working hypotheses on the cause for cell stimulation. Most of these
hypotheses are concerned with the metabolism of the bacteria.
Metabolic Products from Known Substaizces : Thc mctabolic products formed by thc crown-gall organisms from various food materials
have bccn studied in a limited way. The earlier classic work of Smith
(1917) and Smith, Brown, and Townsend (1911) reported analyses
which showcd, in crown-gall bouillon culturcs, thc prcseiice of carbon
dioxide, alcohol, ammonia, amines, fatty acids, acetic acid, formic acid,
aldchyde, and acetone. Sagcn, Riker, and Baldwin ( 1934) and Piiickard
(1935) have reported that several of these substances, such as alcohol,
ammonia, amines, and formic acid, were available under certain conditions to the crown-gall organism. Quantitative determinations of the
products formed by a singlc-cell culture of Phytoinonas tacrnefaciens
from glucosc were undertakcn by Conner, Peterson, and Riker (1934)
in a liquid yeast-infusion mineral-salt medium. The traces of acid
found could be explained by the carbon dioxide present. A fair amount
ATYPICAL AND PATHOLOGICAL MULTIPLICATION OF CELLS
323
of this was given off, but so slowly that it was not detected in ordinary
culture or fermentation tests. Bacterial gum was also produced, as
well as non-reducing, carbon-containing gum-like substances which
were recovered from the cultures in the form of compounds with heavy
metals. These products are probably important in relation to the low
tensiometer readings in giant colonies, discussed later. The consideration of capsular and gum-like materials in relation to pathogenesis
is beyond the scope of this paper. I n addition to these or related substances, hairy-root bacteria ( P . rhixogenes) formed both acetic and
pyruvic acids. The presence of pyruvic acid may perhaps account f o r
some of the results in the analyses reported by Smith. Ammonia was
reported (Wilson, 1935) to be given off by the hairy-root bacteria from
yeast-infusion glucose agar and from carrot-extract agar as well a s
f rom liquid media of similar composition. The crown-gall organism
produced free ammonia from liquid carrot-extract medium, but little o r
none from carrot-extract agar or from yeast-infusion glucose liquid or
solid media. Similar results were secured with a non-pathogenic culture, which indicates no direct correlation between ammonia production
and pathogenicity as suggested by Smith (1917). No toxins in culture
were found (Kauffmann, 1930; Thung, 1929) that had an action on
plants like that of the bacteria. As noted later, crown-gall tissue has
been found commonly to be more alkaline than neighboring healthy
tissue. While further studies on the metabolic products of the bacteria
in relation t o cell stimulation seem promising, other changes also deserve attention.
Changes Iiadzcced i w Culture Media: The hydrogen-ion concentration
of yeast-infusion glucose medium was practically unchanged by crown.*all bacteria. However, in certain other media an acid reaction was
?
induced; in still others, an alkaline reaction, as discussed earlier in
relation to the utilization of various substaiices and to the formation of
metabolic products. The reason for the change was quite clear in some
cases; e.g., the utilization of the nitrogen in ammonium sulfate o r in
sodium nitrate (Sagen, Riker, and Baldwin, 1934), leaving sulfuric acid
or sodium hydroxide, respectively. In tomato decoction (Boehm and
Kopaczewski, 1929 ; Riker, 1926) an alkaline reaction was induced,
perhaps because of (1) the release of ammonia from proteinaceous
materials or their derivatives, and ( 2 ) the utilization of the salts of
organic acids. Wilson (1935) reported that change in the hydrogenion concentration of surface growth on agar varied from that in the
same medium without agar. He found also that the reaction changed
with time, suggesting the successive utilization of diff'erent materials
from the substratum. Pinckard (1935) found that there was characteristic variation in a number of media in the reaction induced, respectively, by several cell-stimulating bacteria (those causing olive knot,
oleander knot, bacterial pocket disease of beets, raspberry cane gall and
crown gall). No direct correlation was found in these cultures between reaction induced and pathogenicity. Changes in p H were also
considered in relation t o changes in Eh.
324
A. J. RIKER AND T. 0. BERGE
The oxidation-reduction potentials in a variety of media inoculated
with Phytornonas turnefaciem have been shown t o become more negative
(Hendrickson, Baldwin, and Riker, 1934 ; Pinckard, 1935 ; Sagen, Rikcr,
and Baldwin, 1934). Similar reduein? actions are common characteristics of other bacterial cultures during growth in artificial culturci
media and therefore are not directly correlated with pathogenicity.
Hendrickson et al. found no significant difference, from the standpoint
of oxidation-reduction potentials induced in ferric ammonium citrate
medium, between crown-gall (both pathogenic and non-pathogenic cultures) and radiobacber cultures. Sagen et al. found that while P. t u m e faciens reduced the potential only slightly in a glucose basal-salt nitrate
medium, there was a decided reduction in the same medium, probably
because of better growth, when 0.2 per cent peptonc was added. Pinckard, growing the organisms mentioned earlier in a liquid glucose mineral-salt medium with varying sources of nitrogen, noticed that the
potential was reduced with ferric ammonium citrate, urea, succinimide,
1-asparagine, 1-tyrosine, 1-cystine, d-glutamic acid, and yeast extract.
Comparable reduction was found in yeast-infusion mineral-salt media
containing a number of different sources of carbon respectively.
Since changes both in hydrogen-ion concentration and in oxidationreduction potentials involve changes in electrical potentials, the question seems worth investigation whether the bacteria have either an
attracting or a repelling effect on various host-cell contents. Evidence
of a n attraction by the bacteria on the nucleus was reported by Riker
(1923b). However, such electrical changes are not the only factors
that might influence the position of the cell contents.
Local changes in surface tension have been regarded by some worliers as important factors in cell stimulation. Chambers (1925) reported
the formation of a surface-tension-lowering. substance by Phytowionas
turnefacierzs in certain media. Arciszewski, Boehm, and Kopaczewski
(1928) and Boelim and Kopaczewski (1929) also found that this organism lowered surface tension in a tomato-juice medium. However,
these investigators observed an increased surf ace tension in bouilloii
cultures. The cause of this change is not understood. It is known,
however, that various substances including acids, esters and aldehydes,
formed by certain bacteria in suitable media, are strong surface-tension
reductants ( Frobisher, 1926). Lower readings with the duNouy tensiometer were obtained by Berge, Riker, and Baldwin (unpublished) in
giant colonies of a pathogenic crown-gall culture on plates poured from
various different kinds of agar than in similar colonies of non-pathogenic sister cultures. Lower readings were also secured in the pathogenic culture at 28" C. than in parallel colonies at 32" C. At this higher
temperature the culture is non-pathogenic on tomato, as discussed later.
Surface-tension changes have been studied by various workers in
relation t o cancers of mail and animals. This subject has been reviewed by Katzenstein and Knake (1931), Malowan (1932) and Traube
(1929), and is beyond the scope of the present paper.
Parallel t o changes in surface tension the total ionic concentration
ATYPICAL AND PATHOLOGICAL M U L T I P L I C A T I O N O F C E L L S
325
was reported by Boehm and Kopaczewski (1929) to be lowered by
P h y t o m o m s tumefaciens in tomato juice and raised in serum. They
found also that the crown-gall bacteria made tomato juice less “stable.”
On the basis of these results, they reported an “antagonism” between
crown-gall bacteria and Streptococcus erysipelatus in respect to several
physical-chemical properties. Inoculations with crown-gall bacteria
followed by injection of the Streptococcus induced only small galls or
none a t all. These studies, in which considerable variability in results
appeared, were made because of the working hypothesis that there
might be an antagonism between the Streptococcus and the unknown
causal agent of cancer.
Changes in osmotic pressure of tissue induced by the crown-gall
bacteria were considered a t some length by Smith (1916b, 1917) and are
discussed later in relation to crown-gall tissue.
The specific enzymes produced by Phytomonas tumef aciens have
apparently received little consideration. The differences in enzyme
content of gall tissue from that of contiguous normal tissue are considered later with the pathological physiology of the host plant. That
the cell-stimulating organisms have a large enzymatic equipment seems
evident from their utilization of a wide range of nutritive materials.
More specifically, tyrosinase was found (Almon and Fred, 1933) in all
of the crown-gall cultures and in 25 per cent of the hairy-root cultures
studied. Klein and Ziese (1932) stated that pure cultures of the crowngall organisms decomposed hydrogen peroxide only to a slight extent.
Doubtless enzymatic action plays a n important r61e in the production
in culture of the metabolic products studied in relation to cell stimulation.
Growth-stimulatiuzg Substauzces
Chemical stimulating agents reported t o induce proliferation in
plant cells include sodium chloride, calcium chloride, ethyl alcohol,
acetone, glycerine, cane sugar, distilled water, extract of kale (Annand,
1927); formamide, formic acid, acetamide (Auler, 1925); 1 per cent
lactic acid (Bittmann, 1925) ; glycine (Hammett, 1934) ; lactic, formic
and succinic acids, asparagine, urea, formalin, alcohol, ammonia water,
tar water, anilin water, arsenic oxide, zinc nitrate, ammonium carbonate, extract from hot pepper, extract from Ricinus, normal and
immune rabbit sera (Kostoff, 1933) ; amber acid, ammonium carbonate,
arsenic oxide, asparagine, f ormalin, formic acid, lactic acid, uranium
oxide, urea, Sofia tap water, zinc nitrate (Kostoff and Kendall, 1933) ;
sodium glycolate (Petri, 1913) ; ammonia water, urea, ammonium acetate, ammonium carbonate, ammonium citrate, ammonium malate, ammonium sulfate, ammonium sulfite, ammonium nitrate, ammonium
lactate, ammonium chlorid, ammonium oxalate, ammonium f ormate,
ammonium salicylate, ammonium succinate, dibasic and monobasic ammonium phosphates ; copper salts, vaseline, paraffin oil, salts of lithium,
excretions from larvae of gall flies, from certain nematodes and from
certain fungi (Smith, 1917); ammonia, acetic acid and formic acid
326
A. J. RIKER A N D T. 0. BERGE
(Smith, 19204. Kendall (1930) injected diastase, pepsin, saliva, asparagin, formic acid, lactic acid, and tartaric acid f o r varying intervals
of time, with little proliferation resulting except, perhaps, in the case
of formic and tartaric acids. Kostoff and Kendall (1933) found that,
in general, the ability of the various chemical agents tested to precipitate plant extracts tended to be directly proportional to their ability
to induce proliferations in Ricinus. Recently, Levine (1934) has
studied the effect on a number of plants of a slight wounding and
subsequent painting with several materials including coal-tar, Scharlach
red, and 1: 2 : 5 : 6-dibenzanthracene. He found that the paintings resulted in a proliferation of tissues about the injured and painted areas
producing swelling of the stems because of the formation of small
irregular masses of new tissue.
Among materials listed as cell-stimulating, glutathione and related
substances contaiiiing the sulphydryl group deserve more attention
than they have yet received. Hammett (1929) called attention t o the
liberation of free sulphydryl from injured root tissue and raised the
question whether Phytomovzas tumefaciem might have a specific type
of sulphur metabolism that could be correlated with its tumor-inducing
properties. He coilsidered that growth by increase in cell numbers
was regulated by an oxidation-reduction equilibrium between compounds containing reduced and partially oxidized -SH groups. Binet
and Magrou (1931) reported that crown gall on geranium contained
much more glutathione than normal leaves o r stems. I n view of the
evidence available, it appears that the “conditioning” effect of such
materials, if nothing more, should be studied in relation to atypical
and pathoIogica1 growths.
A specific bacteriophage, intimately associated with the crown-gall
organism, was reported by d’Herelle and Peyre (1927). Their observations suggested the hypothesis that the true parasite might not be
the normal bacterial form of Phytonaoms tumefaciens, but the filtrable
protobacterial form of this organism induced by the bacteriophage.
They reported that the protobacterial form obtained from gall filtrates
returned to the normal bacterial form. Filtered extracts from crown
galls on sugar beets were capable, in many instances, of inducing similar
galls on other sugar beets, and from these the usual form of the pathogen could be isolated. Chester ( 1 9 3 3 ~ isolated
)
a bacteriophage from
40 per cent of the crown galls induced on Pelargonium stems by inoculation with a strain already containing a weak bacteriophage, from 30
per cent of the adjacent healthy tissues, but from none of the sound,
non-infected plants. In corresponding tests with beet roots, he demonstrated the presence of a bacteriophage active against P.turnefaciens
in a relatively high percentage of non-infected plants. He regarded
this as evidence that a bacteriophage from the soil may penetrate the
healthy root tissues. Coons and Kotila (1925) found that cultures of
the crown-gall organisms to which the lytic principle was added showed
decreased motility, agglutination, and aberrant bacterial forms. They
suggested that lysis may play an important r6le in nature in rendering
ATYPICAL A N D PATHOLOGICAL M U L T I P L I C A T I O N O F CELLS
327
P. tumefacievts avirulent. Israilsky (1927) demonstrated a bacteriophage in gall tissue of beets but not in healthy tissues. After treatment with the bacteriophage he found that P. tzcmefaciews showed a
decreased virulence as evidenced by a lower percentage of successful
inoculations. He stated that the difficulty in culturing the crown-gall
organism in large numbers from gall tissue was owing to the presence
of bacteriophage. Muncie and Pate1 (1929) reported that cultures of
the crown-gall organism after treatment with bacteriophage failed to
infect tomato plants. Brown and Quirk (1929) also found that the bacteriophage reduced the virulence, in varying degrees, of young crowngall cultures. They repeated the d’Herelle and Peyre experiments with
negative results. Kauffmann (19283) was unable to demonstrate any
influence of the bacteriophagous lysin either in tumor production or in
its prevention. Thung (1929) also failed to confirm the work of
d’Herelle and Peyre.
“ Tumef aciens-plastin,” a substance thought by Bechhold and Smith
(1927) to be elaborated by Phytomonas tzcmefacievts in culture media,
was reported to have the property of inducing cell proliferation in
plants, comparable to that induced by inoculation with the crown-gall
organisms themselves. They conceived of this substance, which withstands boiling for twenty minutes and 1 per cent phenol for an hour,
as a non-living, thermostable, filtrable, colloidal or semi-colloidal substance. Other workers (Kauffmann, 19283 ; Thung, 1929), using similar filtrates of the bacterial cultures, have been unable to confirm these
results. Kauffmann (1930) concluded that only living bacterial cultures and not culture filtrates are capable of inducing plant tumors.
Other growth-stimulating substances of unknown composition deserve attention in relation to this complex problem. Smith (1916e)
suggested that endotoxins released only after the death and disintegration of the bacteria might be responsible. However, efforts to
secure stimulation either by dead cultures or by their extracts (Blumenthal and Hirschfeld, 1922 ; Kauffmann, 19283 ; Thung, 1929), comparable to that secured by living cultures, have failed. Cultures of
this organism, like those of many other microorganisms, have been reported (Burrows, 1925, 1926) to contain vitamin B. The rather complete enzymatic equipment of Phytomonas tumefaciens is evident from
the variety of reactions it can induce on various materials. While the
present writers have not observed accounts of this organism producing
bioses, hormones, etc., such reports may perhaps be expected.
While reviewing .this whole question of growth-stimulating materials, one finds pertinent Levine’s (1934) statement that in no case did
the swellings approach the large tumor-like growths induced by inoculations with Phytomonas tumefaciens in plants grown under similar
conditions. Doubtless, this is also true for the other substances listed.
Levine concluded that Smith’s (1917) hypothesis of growth stimulation
by metabolic products formed by P. tumefaciem was inadequate to explain crown-gall formation, and that the presence of the organism in
the tissue may have some function in tumor development other than
328
A. J. RIKER AND T. 0. BERGE
producing the irritating substances. Although there is much to support this criticism, one should hold in mind the inadequacy of the
experimental procedure. It seems very difficult (1) to introduce a
chemical substance into a plant with the same distribution as the
bacteria and (2) to maintain a suitable concentration. However this
may be, many different kinds of substances injected into plants have
been said to induce tumor-like formations resembling in some degree
the crown gall caused by P. turnefaciens. There is, of course, considerable latitude in the degree of resemblance, which obscures the situation. I n any case, the great diversity of materials reported to cause
stimulation suggests that the irritation or injury induced by them, which
is apparently a common characteristic, is more important than the
nature of the substance. I n his comparison between callus formed
after a wound and crown-gall tissue, Riker (1927) said,
. in a
callus the stimulus is comparatively weak, is definitely localized, and
soon disappears; whereas in the gall the stimulus is comparatively
strong or operates over a longer period of time, is diffused within a
limited degree, and persists. It is not inconceivable that the fundamental stimuli may be somewhat similar in both cases.”
But whatever the bacterial factor for stimulation may be, it is unable
to induce proliferation in a resistant plant.
“. .
Resistance and “Avztibodies”
The apparent immunity to crown gall of cektain begonias, as well
as of certain other plants, was correlated by Smith and Quirk (1926)
with hydrogen-ion concentration. They showed that these plants had
a reaction more acid than pH 5.0, the limit of crown-gall toleration in
bouillon. Riker et al. (1930) reported that 4.4 was the limiting pH for
initiating growth in a yeast-infusion mineral-salts medium. Muncie
(1930), who reported the acid tolerance in peptone bouillon as p H 5.8,
observed the presence of crown galls under natural conditions and ready
gall production by inoculation with Phytomonas tumefaciens in the very
acid Rumea crispus (pH of leaves 4.8; roots, 5.2; gall on roots 5.9)
and also in Rheum rhaponticzlm (pH of shoot 3.7; rhizome, 5.5; gall,
5.7). He showed that even in these hosts the crown-gall organism was
able to bring about a favorable condition for the initiation of gall
development. While doubtless the amount of inoculum introduced and
other local conditions may influence the ability of the bacteria to become
established and to induce infection, it seems reasonable that a strong
acid reaction in plant tissue might increase the difficulty of infection
by crown-gall bacteria. This question of acidity also affects agglutination.
Acid agglutination of Phytomovzas tzlmefaciens was reported by
Soru (1929) to take place actively between pH 5.7 and 6.9. The agglutination and plasmolysis of the crown-gall organism by fresh potato
extract have been reported (Berridge, 1929) to depend on the hydrogenion concentration of potato tissue itself. Korinek (1924) observed
ATYPICAL AND PATHOLOGICAL MULTIPLICATION O F CELLS
329
marked aggregations of the crown-gall organism in the juice of B e t a
vulgaris, but regarded them as phenomena of absorption and chemotaxis
about crystals found in the sap, and not as true agglutination. The
evidence on agglutination appears inadequate as yet to deserve much
consideration in relation to resistance.
The possible production of immunity in plants following inoculation
with crown-gall organisms has attracted the attention of various workers, some of whom have reported apparently successful artificial immunization in plants. Among the positive reports (Arnaudi, 1926,
1927, 1928) it appeared that, when geranium shoots were inoculated
with Phytornoszas turnefaciens 3 to 4 em. above existing tumors induced
by the same organism, no new tumors or only occasional small tumors
were induced. Parallel control plants developed crown galls in 6 weeks.
When tumor-bearing Pelargonium branches were cut off and placed in
a 1per cent solution of antiserum from immunized rabbits, regression
of the tumors occurred within one month. Tumors on control branches
placed in sterile water remained green, turgid, and viable for two
months. Branches placed in the serum solution immediately after
inoculation failed to produce galls, while controls in pure water did so
readily (Arnaudi, 1927). GFheorghiu (1932) reported ( 1 ) immunity to
crown gall in Pelargonium thirty to forty days after the introduction
of a large quantity of an emulsion of heat-killed crown-gall organisms,
and ( 2 ) the cure of existing galls by repeated swabbing of exposed
tissues with a similar vaccine at intervals of five to six days. Smith,
Brown, and Townsend (1911) showed a tendency for cuttings from
daisy strains which had borne crown galls to become resistant to P.
turnefacieszs. On the other hand, later work led Smith (1916e) to state
that whatever immunity was induced in the plant appeared to be temporary. Brown (1923) found a slight temporary resistance in several
cases of daisy cuttings and rose seedlings, but this resistance was lost
and increased susceptibility to the crown-gall disease was noticed after
several generations. She found that when dead cultures of P. tumefaciens were injected into the vascular bundles of Paris daisy plants,
some degree of immunity developed if virulent cultures were inoculated
in the spot where the dead cultures were injected. However, galls
developed in each case when the crown-gall organism was inoculated in
the internode above the injected area. Therefore, the results indicate
that if antibodies are developed, they are not carried through the sap
of the host so as to induce an effect in some other portion of the plant.
No significant difference was found (Riker, 1926) between galls on
control tomato stems and those on stems with previously well developed
galls. Thung (1929) failed to induce immunization of Riciszus commumis against P. tumefaciens by means of a vaccine prepared from the
organism. Chester ( 1 9 3 3 4 suggested that the diffusion of a bacteriophage for a short distance around the site of injection of crown-gall
bacteria might explain, by its prophylactic value in healthy plant tissues, results like those reported by Arnaudi in the vaccination of plants
against crown gall. From the studies thus far made, it appears that
330
A.
J. RIKER AND T.
0. BERQE
evidence in favor of immunity in plants t o crown gall following inoculation is not satisfactory. Among several factors bearing on this
subject, one particularly, noted by Smith, Brown, and Townsend (1911)
and others, seems to have been overlooked by some workers who reported immunity; ‘uix., galls develop satisfactorily only on well growing
plants. Consequently, if a stem was somewhat stunted by a previous
treatment, it might have the appearance of resistance t o subsequent
inoculation because of reduced rate of growth. This question is treated
more completely on a later page.
The evidence on antibodies against Phytomonas tumefaciens in
plant tissues infected with the crown-gall organism is largely negative.
Arnaudi (1927), Carbone (1926), Korinek (1924), and Riker ( 1926)
were unable to demonstrate the presence of precipitins or agglutinins
in the host tissue of tomato, geranium, or beet plants, near galls or in
the galls proper. However, Magrou (1935) found one type of agglutinating material in juice from crown gall of geranium and of Paris
daisy, but a different type in that from healthy tissue of the same
plants. Similar trials with beets were negative. H e also found material in the juice from crown galls, but not from healthy tissue, that
formed a precipitate with cultures of P. tumefacicns and four other
unrelated bacteria pathogenic on plants. Although detectable antibodies following injection of crown-gall bacteria have not been reported
commonly in plants, they are easily induced in animals. Riker (1926)
prepared rabbit serum which agglutinated P. tumefaciens in a dilution
of 1 to 3000. Link and his associates (1927, 1928) and Pate1 (1929)
also induced antibodies in rabbits against crown-gall organisms and
other plant pathogens in their series of cross-agglutination studies.
The “precipitins” found by Kostoff (1929) in grafted plants which
developed spontaneous tumors and said t o result from a physiologic
interaction between the constituent elements specific to scion and stock
respectively, have been shown by others (including Chester and Whitaker, 1933; Whitaker and Chester, 1933) to result from reactions between calcium and oxalate ions in the majority of cases in the plants
used by Kostoff. I n the other positive results, reactions were caused
by combinations of two unknown substances, one probably a protein,
and the other a less complex organic substance. I n some plants, not
used by Kostoff, still other substances were found by Chester and
Whitaker (1933) to be responsible f o r precipitin reactions. However,
none of these reactions could be attributed to the presence of true
antibodies. While repeating Kostoff ’s experiments on acquired immunity in the Solanaceae, Wilhelm (1933) confirmed in a general way
Kostoff ’s data on the precipitation capacity of the resultant extracts,
but could gain no evidence of an enhanced precipitation over that from
grafted shoots. Kostoff’s interpretations were opposed by SilberSchmidt (1933).
The general question of antibodies in plants is reviewed by Chester
(1933b).
ATYPICAL AND PATHOLOGICAL MULTIPLICATION O F CELLS
331
Dissociation
Morphological variations of the crown-gall organism have been
observed by various workers. Smith, Brown, and Townsend (1911)
found that Phytomonas tarmefaciens varied in size and readily developed involution forms under unfavorable cultural conditions. Threemonths-old cultures of the organism grown on bean agar and stained
with Loeffler’s methylene blue were shown by Levine (1925d) to consist
of an amorphous mass of jelly-like substance with occasional deeply
staining, minute, spherical bodies. Transfers of the culture to the
same medium gave rise to long, beaded rods, which gave way, in turn,
to small, faintly staining cocci with occasional slender bacillary forms
or filaments as the age of the culture increased. After a certain length
of time, the subculture was found to resemble the original culture.
Levine found in these observations support for Lohnis’ (1921) studies
on the life-cycle of bacteria. Small lenticular bodies equal in length
to P. tumefaciens, but somewhat wider, were also observed. Both ends
of this body took deep stains. The hyaline central portion failed to
stain and was regarded as a true spore. Rosen (1926) opposed Levine
on the formation of spores by the crown-gall organism on the basis both
of staining reactions and of thermal relationships, since the lethal temperature f o r this organism is quite low (Rosen, 1926; Smith, Brown, and
Townsend, 1911; Walkden, 1921). Doubtless these workers had different concepts in mind when they employed the word “spore.” The
various sizes ascribed to the crown-gall organism by Riker (19234 and
Robinson and Walkden (1923) were considered by Levine to result
from different ages of the cultures used. I n accord with earlier reports
and the usual situation (Henrici, 1928) in bacteria, Rosen (1926) found
a marked variation in size and shape of the rods in young, vigorously
growing cultures of P. tumefaciens. He observed club-shaped, bipolar
staining, and apparently budding organisms, the last suggesting multiplication resembling that in yeast cells. Although he reported filterpassing forms of the crown-gall organism, he found in the filtrates
certain contaminating bacteria which cast doubt on the experiment.
Changes in colony characteristics of the crown-gall organism on
solid media have been observed. Israilsky and Starygin (1930) obtained from ordinary cultures mixtures of smooth and rough colonies,
the two forms showing different cultural characteristics in milk and in
certain carbohydrate media. Quirk (1931) stated that pure cultures
of smooth o r rough colonies could be obtained at will in beef-infusion
broth by varying the hydrogen-ion concentration of the medium. Link
and his associates (1927, 1928) observed colonies intermediate between
S and R occurring naturally in culture media. These colonies tended to
become entirely smooth after several days. Wright, Hendrickson and
Riker (1930) pointed out the difficulty of securing pure cultures of
the crown-gall bacteria and their necessity in studies on variability of
single-cell cultures. However, both smooth and rough colonies of
Phytomonas tumefaciens have been f ouiid even in single-cell cultures
332
A. J. RIKER AND T. 0. BERGE
(Hendrickson, Baldwin, and Riker, 1934). Because of various evidence, including loss of pathogenicity by certain strains, Brown and
Leonard (1932) suggested that the crown-gall organism might be a
mutant, a physiological form, or one of the pleomorphic forms of
Bacillus radiobacter. This relationship is considered in detail by
Hendrickson, Baldwin, and Riker (1934).
Dissociation in the extreme was discussed by Lieske (1928). He
presented the bold idea that the various organisms claimed by different
workers as specific causative agents of cancer were all readily demonstrable dissociated forms of Phytomolnas tzcmefaciews. His translated
statement (pp. 395-396) follows : “My own extensive examinations now
yielded the following conclusions: 1. Cancer is caused by a specific
agent. 2. The statements of Gye and Barnard and many other investigators that the cancer agent is filtrable is correct and easy to prove.
3. The Gram-negative bacterium isolated from cancers by Blumenthal
and his co-workers, as well as by other investigators, is identical with
the agent of plant cancer. The deviations of the individually isolated
strains lie within the range of variation of this organism. Different
types of bacteria are not involved, as, f o r example, Reichert asserts.
Bacterium tzimefacierzs is the specific, true, and only cancer agent in
plants, animals, and man. It is not, as is often assumed, to be considered in the same category with the most diverse other causes of
irritation. 4. The Gram-positive, spore-forming bacilli isolated from
cancers by Bins and Riith, Glover, Young, and others, may be demonstrated in every true cancer; they are the typical, true, and only cancer
agents. 5 . The streptococcus first described by Nuzum is actually, as
Americans maintain, ‘the ultimate cause of cancer. ’ I was able to find
it in 100 per cent of all cancers.” I n brief, he considered that the
crown-gall organism, through its dissociated forms, was the true causative agent of cancer in plants, animals, and man. However, a number
of obstacles, as considered elsewhere, oppose his concept. They include the following: (1) There is insufficient evidence of a causal connection between any organism (excepting, perhaps, certain viruses)
and animal cancer. (2) The relations between organisms associated
with animal cancer and P. tumefacierzs are not satisfactorily proved.
( 3 ) The pathogenicity of crown-gall organisms for warm-blooded animals has not been demonstrated,
I n connection with these studies on variability and dissociation of
Phytomolnas tumefacielns, it seems desirable to emphasize the importance of single-cell cultures. Then, after every precaution has been
taken against contamination, there are many pitfalls for even the well
trained investigator (Zinsser, 1932). It appears that, so far as the
crown-gall organism is concerned, the evidence is not satisfying for
dissociation beyond the appearance of rough and smooth colonies, the
formation of gonidia, and certain other changes (e.g., Hendrickson,
et al., 1934) in single-cell cultures.
ATYPICAL AND PATHOLOGICAL MULTIPLICATION O F CELLS
333
Radiatiort
The problem of inducing variation in the host plant by radiation
(1) from the bacteria, (2) from radium, or (3) from x-rays will be
considered before passing to the physiology of the galls proper.
“Mitogenetic radiation” in relation to growth stimulation by crowngall bacteria has been given attention by Magrou. He found (1928,
1930) that the bacteria increased the number of mitotic figures in onion
root tips, not only directly but also at a distance and through a quartz
barrier. Abnormal development of fertilized eggs of Paracewtrotus
lividus Mrtsn. was induced by a forty-eight-hour exposure. The fact
that the effect passed through quartz and water, but was stopped by
glass o r gelatine, opposed Choucroun’s (1931) objection that the result
came from leaky apparatus. It was attributed to short rays from 2,370
t o 1,990 A. Since physical measurements were negative, it was impossible “formally” to conclude that short rays were the cause, although
they provided the best explanation. Magrou (1930) observed that the
oxidation of glucose gave similar results. Choucroun (1931) secured
comparable effects with a non-pathogenic yeast and bacterium. Certain
phosphorescent bacteria and also Phytonzonas turnefaciens stimulated
mitosis when contained in a quartz tube inserted in the marrow of the
rabbit tibia (Soru and Brauner, 1933). The results (with 12 animals)
were quite variable. Thung (1929), supporting Magrou, considered
that mitogenetic rays were responsible for crown-gall development.
These results perhaps deserve consideration in relatioa t o other lines
of work. (1)The onion has been considered (Smith and Quirk, 1926)
as not easily, if at all, infected by the crown-gall bacteria. (2) The
existence of mitogenetic rays is still undecided, since both positive and
negative results have come from reputable laboratories (Duggar, 1935).
Radiation emitted by the crown-gall organism was considered by
Lakhovsky (1929) to be responsible for abnormal cell proliferation
because of disturbances in the “oscillatory equilibrium’’ of the normal
cell. He thought that if these disturbing radiations were neutralized
the galls could be prevented from development. He reported curing
crown galls on Pelargonium by negating the effect of the mitogenetic
radiation with a copper spiral, isolated by ebony, and placed around
the diseased plants at the level of the galls. Various workers (Rivera,
1928b; Stapp, 1933; Stapp and Bortels, 1933) have repeated this experiment. Rivera approved of the idea, but the others did not. None
secured Lakhovsky’s results. Although the crown-gall bacteria may
have an electrical influence upon the host tissue, as mentioned in studies
of hydrogen-ion concentrations and oxidation-reduction potentials, the
available evidence in favor of effects by radiations from the bacteria
seems defective.
Radium emanation tubes inserted into crown-gall tissue inhibited
its development as shown by reduction in tumor size (Levin and Levine,
1922). Likewise, the insertion into geranium stems of capillary tubes
containing 0.3 to 0.6 me. of radium, within twenty-four to forty-eight
334
A. J. RIHER AND T. 0. BERGE
hours after inoculation, was found (Levine, 1925c) to inhibit the development of crown galls. Levine considered this fact to be significant,
since Riker ( 1 9 2 3 ~ )had demonstrated that the application of a hot
needle into the wound, half an hour after inoculation, did not stop gall
formation. Levine also found that death of crown galls occurred after
certain amoullts of unfiltered irradiation from radium. The visible
microscopic effects of such emanations appeared to be caustic in nature.
Although the crown galls were found to regress and die following this
treatment, they often grew again from the margins of treated areas.
Rivera reported ( 1 9 2 9 ~ )curing plant tumors with radium, but found
(19284 that very strong doses of gamma rays were unable to kill agar
cultures of P. tumefacierzs. The development of the organisms v a s
delayed during and as a result of the treatment. The alpha and beta
rays were absorbed by the walls of the tube. From further studies
(1929) on directly exposed cultures he concluded that the gamma rays
inhibited the growth of the bacteria which were then killed by the beta
rays.' He showed also (1929a) in higher plants that radium emanations provoked a rest period-a cessation of cell multiplication-even
in those plants which normally show none. Tumors on Ricinus were
killed by an initial dose of 13 microcuries but normal tissue was also
injured (19293). Arnaudi and Venturelli (1934), using radio-activated
water containing 1,600 units Mache of emanation equivalent to 640
microcuries, and direct exposure to radium filtered with 5 mm. of lead
and 1.5 cm. of Columbia paste, secured positive results on Ricirzus communis inoculated with P. tumefaciem.
Similarly, x-ray treatments under certain conditions have been reported to exert a specific influence against the diseased cells of crown
gall while leaving unchanged the normal tissue of mature plants. Rivera (1926) stated that small x-ray doses stimulated the growth of
tumors on Ricinus and Pelargonium, at first, but subsequently these
tumors died much more rapidly than those untreated. With larger
doses, roentgen rays were capable of arresting the development and
of bringing about a regression of galls induced by Phytomorzas tumefacierzs. Irradiation with unfiltered rays brought about regression of
galls after a preliminary period during which the galls continued to
grow by increase in cell size (1925, 1927). Histological examination
of the tumors showed that the x-rays caused disorder of the nuclei and
enlargement of the cells with an increase in cytoplasm to a point where
the cell walls were ruptured and death of the tumor resulted. Cell
multiplication was apparently checked through inhibition of nuclear
divisions. The same treatments were without effect on mature healthy
tissues if not applied more than twice. Likewise, cultures of P. tumefacierzs were uninjured on agar (1925) or in broth (19273). However,
negative results were obtained with x-ray treatments (including 600 r,
90 kv., no filter for thirty minutes) in two separate studies (Stapp,
1933; Stapp and Bortels, 1933) that were designed to repeat Rivera's
work. Such results suggest withholding acceptance of the positive
results reported untiI the evidence in their favor is improved. Komuro
ATYPICAL AND PATHOLOGICAL MULTIPLICATION O F CELLS
335
(1924) observed degenerative phenomena and cytological changes in
seeds, seedlings, and young plants of Vicia faba upon exposure to xrays. He concluded that x-irradiation of Vicia faba leads the cells
of the vigorous growing point to a diseased or senescent condition
resembling that of malignant tumor cells and then t o death. Stein
(1932) likewise observed degenerative alterations in embryonal and
post-embryonal tissues in an Antirrhinum embryo treated with beta
rays of radium. A chromosomal mutation was found to be involved,
since the changes were shown through four generations. She stated
that the heredity of the “disease” was complex. Several hereditary
factors were involved, one of which was recessive. I n view of the working hypothesis that tumor cells may be mutants from normal cells, and
the demonstrations that mutations follow radium and x-ray treatments,
it appears that such treatments should be useful tools f o r study in this
direction.
Hertzian waves (2 meter lengths at 40 watts with 10 em. distance
for 100 hours during twelve days) were found (Rivera, 1931) to stimulate new tumors, but old tumors ceased to grow. Gosset, Gutmann,
Lakhovsky, and Magrou (1924) reported that crown gall on Pelargonium was cured by treatments with waves 2 meters long having a
frequency of 150 million per second.
The cures of crown gall with irradiation raise the question concerning how much of the effect may be accounted for by reduced growth of
the host plant.
Since the general question of irradiation on plants is reviewed by
B, M. Duggar (1935), further consideration of this subject is omitted
in favor of reports on other physiological studies.
Physiology of the Host Plant
The development of crown gall is commonly correlated with that
of the host, as observed first by Smith, Brown, and Townsend (1911)
and later by a number of writers. Levine (1921) and Smith (1928)
found that a well nourished, vigorously growing host responded to the
invasion of Phytomoizas tumefacierzs t o a greater extent than a poorly
nourished or feebly growing host. Levine indicated (1923) that the
size of the gall depended much more on the vitality of the host and the
region of inoculation than on the size of the inoculum. Flowering and
fruiting in Pelargovlium xonale and Datwa stramoizium were found by
Stapp and Bortels (1931) distinctly to inhibit tumor formation. F o r
example, the fresh weight in grams of crown galls formed by single
inoculations of the crown-gall organism on fifteen flowering thorn-apple
hosts was 10.6 g., while crown galls on the same number of nonflowering plants weighed 42.4 g. Similar but less clear-cut tendencies
were noticed also in tomato plants. Riker and Hildebrand (1934)
demonstrated that inoculations of P. tumefacierzs in apple trees which
\yere making little or no growth did not induce the disease until the
trees began to develop. The reactions on such trees were often nega-
336
A. J. RIKER AND T. 0. BERGE
tive. Sometimes development of the crown galls appeared after a
normal incubation period dated from the beginning of the growth of
the tree. Riker (1926) pointed out that there was a correlation between the development of the gall and the growth of tomato i n temperature and moisture studies.
The influence of temperature and soil moisture on the development
of crown gall and the tomato plant was examined (Riker, 1926) in
Wisconsin temperature tanks and chambers. When temperature tanks
were used, it was found that a temperature of 30" C. and a soil containing 80 per cent of its moisture-holding capacity favored the maximum development of tomato plants. The optimum temperature for the
development of galls, however, was about 22" C., and the optimum
moisture about 60 per cent of the moisture-holding capacity of the soil.
The minimum and maximum temperatures for the development of galls
were approximately 10" and 30" C., respectively; and f o r tomato plants
approximately 10" and 37" C. Galls probably failed to develop a t low
temperatures because of poor growth by the host plant. A series of
experiments on tomato plants in a more closely controlled environment
was made in chambers with air temperatures between 24" and 36" C.,
and with a relative humidity about 70 per cent saturation. Good development of galls was secured between 24" and 28" C. Between 28"
and 30" C. the galls were distinctly inferior, while above 31" C. no galls
were induced, although the plants grew well a t 32" C. Maximum growth
of the crown-gall organism, as measured after ten days by colony size
on nutrient-dextrose agar, occurred between 18" and 30" C. with the
best growth a t 22" C. I n tomato extract, measurements of hydrogenion concentration showed active-growth range of 18" to 34" C., with
the greatest alkalinity at 30" C. Measurements of turbidity in a liquid
asparagine glucose medium showed a similar active-growth range. A
slight growth appeared at 6" C. On yeast-extract glucose agar a t p H
7.0, maximal colony size was found (Pinclrard, 1935) after fourteen
days' incubation at 28" C., with good growth also occurring at 36" C.
Thus it appears that galls were inhibited from growth at 28" to 30" C.,
a lower temperature than that having much effect on the development
of either host or parasite. These considerations make it appear that
there is a marked change in the physiology either of host or of parasite,
or of both, at 28" to 30" C., which may be correlated with pathogenicity.
Similar work on other hosts is needed.
Chemical differences between crown-gall tissue and normal tissue
have been observed by various workers. The sugar content has been
reported to be considerably decreased in galls of sugar beet ( Townsend,
1915; Strohmer and Stift, 1892). Kostoff (1933) noticed an accumulation of starch in galls, Proteinaceous substances were greatly increased in gall tissue of balsam, geranium, tomato, turnip, and beet
according to Klein and Keyssner (1932a). They found an increase in
soluble inorganic and organic nitrogenous compounds (ammonia, aminoacids, amides) in the tumor tissue of balsam, geranium and tomato,
but a decrease i n these substances in the tumor tissue of beets, These
ATYPICAL A N D PATHOLOGICAL M U L T I P L I C A T I O N O F C E L L S
337
investigators (1932b) found the soluble acids including alkali-bound
acids in the geranium and tomato galls lower than in the stems. The
ash contents and their alkalinities were determined on geranium, tomato, turnip, and beet. On the basis of fresh material the gall tissue
was more acid (like results of Smith, 1925; and Brown and Quirk,
l929), but on the basis of dry substance it was more alkaline and parallel
t o the p H values. An increased tannin content in galls o n apple was
reported by Sylwester and Countryman (1933). Riker (1923~1and
1927) noted deposits of granules of pectic substance, calcium oxalate
crystals, and various unidentified materials in gall tissue of tomato
plants. Milovidov (1930) reported tannin. Glutathione was found in
considerably larger quantities in tumor tissue of Pelargonium than in
the normal tissue of the same plant (Binet and Magrou, 1931). It
seems desirable in continuation of studies of this kind that differentiation should be made, if possible, between the activity of the bacteria
and the corresponding response of the host tissue. A number of the
materials mentioned above are used by the bacteria, as already considered in an earlier section. I n addition t o differences between gall
and healthy tissue in regard t o materials present, various other important differences between them have also been studied.
The hydrogen-ion concentration of gall tissue has been found to be
somewhat more alkaline than that of normal tissue by a number of
workers (including Brown and Quirk, 1929 ; Harvey, 1920 ; Klein and
Keyssner, 193221; Muncie, 1930; and Smith, 1925). F o r example,
Muncie reported p H values in Rurnelr; crispzcs to be: in leaves, 4.8, in
healthy roots, 5.2, and in gall tissue of roots, 5.9. I n Rheum rhaporhticum p H values ranged from p H 3.7 in the shoot and pH 5.5 in the
rhizome to pH 5.7 in gall tissue. Brown and Quirk found the relationship reversed after oxidation of gall tissue. Whereas the juice of the
normal sugar beet immediately after crushing might be pH 5.9, and
that of the crushed tumor 6.3, in two days the juice of the normal sugar
beet would be p H 6.2 and that of the tumor 4.8. However, Smith (1925)
and Brown and Quirk (1929) reported the titratable acidity in gall
tissue to be considerably greater than that in normal tissue. Klein and
Keyssner ( 193221) made investigations on balsam, geranium, tomato,
sunflower, cucumber, turnip, and beet. I n all cases the galls were more
alkaline in reaction than the neighboring healthy tissue. Even small
tumors showed an alkaline reaction in comparison with the contiguous
stem. Wilson’s (1935) confirmation of earlier reports on the production of ammonia by the crown-gall organism and his finding it produced
from plant materials are particularly pertinent in this connection. With
a different technic, Berridge (1930) stained free-hand sections of crown
galls on sweet pea with methyl or diethyl red and showed that while
the phloem and parenchyma gave a reaction of approximately p H 5.8,
the intercellular bacterial zoogloeae were considerably more acid (about
pH 5.0). A red coloration of the walls of the cells in gall tissue approximating the p H of the bacterial zoogloea was also noticed, being
most marked in the middle lamella. This does not correlate easily with
338
A. J. RIKER AND T. 0. BEROE
evidence dealing either with cell sap or with the reaction induced by
the bacteria from known materials. It suggests the desirability of
further examination of the condition immediately surrounding the bacteria in the tissue. Doubtless, studies which differentiate more clearly
between bacterial action and host response will be helpful.
The influence of reaction on respiratory enzymes associated with
growth was considered by Harvey (1920). He reported that “ a change
from pH 5.52, the average value for healthy stem tissue of Ricinus, to
pH 5.78, the average value for tumor tissue, increases the catalase activity 25 per cent at an acetate concentration of fiftieth normal. The
activity of oxidase is increased also by a decrease in the H
concentratioii.” However, Klein and Ziese (1932) reported that differences
in hydrogen-ion concentration were not great enough to explain the
differences in catalase activity between tumor and healthy tissue of
beets, geranium, sunflower, sedum, tomato, and balsam. The diff erence
in enzyme activity is considered later.
The oxidation-reduction potential is doubtless important in gall
development. As noted earlier, this potential is reduced by growth
of the crown-gall organism in culture. However, in plant tissues Soru
(1931) reported that the tumor tissue was more positive than the
healthy tissue. Brown and Quirk (1929) found that freshly extracted
tumor juice from crown galls on Ricinus and sugar beet was always
more negative than normal juice. This relationship was reversed on
standing, doubtless because of further oxidation.
The depression of normal cell respiration has been advanced as a
cause of crown-gall formation in plants by Smith ( 1 9 2 0 ~ ) . According
to Smith’s hypothesis, the metabolic products of the crown-gall organism acted by causing cell precipitates which upset respiration and
compelled the cells to rearrange their protoplasm and to divide if they
were to live. He stated (1925) that “ oxygen-hunger )’ was the result
of disturbed respiration caused by extraneous substances thrown into
the tissues as the by-products of the parasite. The next step was coiiceived to be an alteration of the surface layer of the protoplasm (ectoplasm) governing outflow and intake, “ leading to loss of solutes and
t o a n excessive intake of water followed by repeated cell division.”
Robinson (1927) mentioned that in many instances in plants, a lack
of oxygen f o r a time resulted in cell proliferation. He saw, as of some
significance in crown gall, that Phytonzoulas tumefaciens was a strongly
aerobic organism, and considered that in the intercellular spaces it
might modify the oxygen relations of the cells so that the internal
changes resulting in proliferation were set up. The decrease of thc
oxidation-reduction potential by crown-gall bacteria has been previously
discussed.
Osmotic pressure changes induced by the crown-gall bacteria, and
the responses of the host tissue to the changes, present some interesting
considerations. Smith (1916b, 1917) suggested that locally increased
osmotic pressures would cause a movement of water and foodstuffs
into the affected area, thus providing excessive nutrient material in a
+
ATYPICAL AND PATHOLOGICAL MULTIPLICATION O F CELLS
339
limited region. Ammonia produced by Phytomonas tumefaciem was
regarded (1917) as “the sole determining factor in abnormal cell proliferation, since as a diffusible stimulant, it would enter cells readily,
would increase the osmotic pressure, and would tend to enter into soapy
combinations with the lipoids of the cell surface, thus changing the
surface tension beyond the point of physiological cell restraint, whereupon cell division would take place. At the same time that ammonia
and other concentrated bacterial products move outward toward the
surface of the tumor, where usually growth is most abundant, water
and dissolved foodstuffs would move inward into the tumor, thus supplying copiously, especially at the periphery of the tumor, both the
necessary stimulus and the substance needed f o r the constantly increasing normal growth.” I n line with this, Kostoff (1933) reported
storage of an abundant food supply of proteins and carbohydrates in
the expanded tumor cells. Strohmer and Stift (1892) found more
water in the gall tissue of sugar beets than in normal tissues. On the
other hand, Harvey (1920) made measurements on expressed normal
liquid and crown-gall liquid from castor beans. More accurate measurements with thermocouples of freezing points of the tissue were reported for a few cases. He concluded that because the differences were
not significant in his limited trials, osmotic pressure relations could not
account for gall production in these cases. However, it appears that
this question should not be lightly dismissed. It seems unlikely that
the organisms would grow in tissue without producing some osmotic
change. Several considerations such as (1) the distribution of the
bacteria within the intercellular spaces; (2) their failure to form conspicuous cavities between the cells ; (3) the appearance of hypertrophic
cells about their location; and (4) their formation of readily diffused
products from certain materials used, raise the question whether the
osmotic pressure of the small bacterial mass between the host cells in
the early stages of gall formation may be lower than that of the contiguous cells. Perhaps light may be shed on this question by experiments which separate the effect of the bacteria from the response of
the host tissue.
Cytoplasmic viscosity increased progressively in tumors on hybrid
plants (Kostoff, 1 9 3 0 ~ )from the peripheral parenchymatous region
toward the central nutritive zone. I n callus tissue, the cells which were
closer to the callus plane had a higher cytoplasmic viscosity than more
distant ones (1930b). Cytoplasmic viscosity was thought t o be of importance in normal cell division (Kostoff, 1930a). While the density
of the nucleus during the resting stage was greater than that of the
cytoplasm, during the prophase and diakinesis the density of the cytoplasm was greater than that of the nucleus. The cytoplasm appeared
to be least viscous during the division phase of the metaphase. Because of the higher cytoplasmic viscosity during the prophase, Kostoff
(1931) assumed that a slight increase in cytoplasmic viscosity brought
about favorable conditions f o r cell division. This explanation was more
plausible since he found (1930d) that spontaneous tumors on certain
340
A. J. RIKER AND T. 0. BERGE
hybrid tobacco plants were readily induced by any agent (wounding,
chemical substances, etc.) which brought about an increase in cytoplasmic viscosity by ‘(despecification” of proteins. I n crown galls, he
considered that the same conditions were brought about by a combination of the action of metabolic products of the bacteria and the abnormal
products of the host cells in affected regions. He found (1933) a positive correlation between chemicals which precipitated plant extracts
and the ability of these chemicals to induce cell proliferation in Ricinus.
In case further studies should substantiate these reports, the question
remains of defining the cause for this change in viscosity.
Enzymatic activity of certain kinds is apparently increased in gall
tissue. Smith, Brown, and Townsend (1911) reported the oxidizing
power of extracts from crown galls on almond and sugar beet to be
greater than that of extracts from sound tissues. Various other workers have pointed out that both catalase and oxidase activity were increased in gall tissue over that in normal tissue (Harvey, 1920; Israilsky, 1927; Klein and Ziese, 1932, 1933). The catalase effect was
stated by Klein and Ziese (1932) to depend on the host tissue or on its
altered metabolism and not on the metabolism of the pathogen, since
pure cultures of Phyt omonas tumefaciews decomposed hydrogen peroxide only to a slight extent. These workers (1933) considered that
there was no diffusion of peroxydase action in tumors of horse-radish,
since the activity of the normal portion of a tumor-bearing root was
the same as that of the root of a normal plant. Riker and Keitt (1926)
found much more oxidase in crown galls than in callus knots, Increased catalase activity has been shown to be a specific phenomenon of
tumor tissue and not of wound tissue (Klein and Ziese, 1932). Catalase was reported to act as an inhibitor of oxidase, and, therefore, a
regulator of oxidation (Bristol, 1916; Lantz, 1927). If the increase in
oxidase activity is proportionally greater than the increase in catalase
activity, the result might be an excessive local oxidation leading to
increased growth in the area affected. Further studies of these questions should include quantitative determinations. I n this connection
one should consider the suggestion that, once started, the crown-gall
cells might continue growth, without continued bacterial action, and
merely under the influence of their own disturbed metabolism.
The question of autonomous growth of tissue cells in crown galls
seems quite important and has been raised because of the failure of
certain investigators to demonstrate the presence of Phytomorzas tumefaciem in older galls either by cultural or by microscopic methods,
This failure with spontaneous tumors from mangels and sugar beets
together with successful isolations from inoculated material led Jensen
(1918) to assume that the abnormal growth of the cells in the affected
region is independent, i.e., without continued relation to the bacteria.
It appeared to him “extremely probable that the cells of the tissue,
under the influence of the bacterium, become for a series of cell-generations altered, and develop the abnormally increased proliferative power
of tumor-cells proper, which is independent of continued stimulation. 7 ’
ATYPICAL AND PATHOLOGICAL MULTIPLICATION O F CELLS
341
Levin and Levine (1920) were led to the same general conclusion, but
for a different reason. They observed sometimes that, when similar
plants received identical inoculations and treatments, some plants had
small harmless galls while others showed large lethal tumors. T o
explain this phenomenon, they suggested that the initial cell proliferation began as a protective reaction of the host against the bacterial
invasion, and ceased with the benign tumor when the organisms were
rendered harmless. But the plant with the lethal tumor apparently
received an impetus for limitless proliferation. E. F. Smith (1922b)
maintained that in tobacco, normal cortex cells became tumor cells upon
contact with the latter, i e . , by appositional growth. He suggested (p.
36) that : “We may conceive of the stimulus as a chemical-physical one
derived from the bacteria and acting either at a distance from them,
i e . , on cells in which they are not present, or a s due to a direct transfer
of the bacteria from cell to cell, the adjacent walls having numerous
very thin places (pits) through which such a transfer might easily take
place by solution of the very thin membrane, or by its rupture due t o
pressure, in which latter case the chemical-physical stimulus would be
confined to the parasitized cells o r at least would not extend beyond their
immediate vicinity.’’ Against the conclusions of Jensen and of Levin
and Levine may be opposed the objection that there has yet to be demonstrated the absence of a continued relation between P. tumefacierw in
gall tissue and continued crown-gall proliferation. Perhaps the nearest
approach to this is the development of callus tissue above a parasitic
overgrowth (Riker and Hildebrand, 1934) in a manner similar to that
above an injury. As has been stated in a previous section, other workers using differential media have been able consistently to isolate virulent organisms from old crown-gall tissue, although small pieces of
tissue could be secured which apparently contained no bacteria.
The possibility of turning malignant tumors into benign tumors has
already been mentioned under sections dealing with radiation and
antibodies. Spontaneous healing has been observed by several workers including Gosset et al. (1934), Hamdi (1932) and Levin and Levine
(1920). Reports of successful work with chemicals have recently
appeared .
Chemical treatment and subsequent necrosis of crown galls without
harmful effects on the plants were reported by Gosset, Magrou, and
Tchakirian (1934). Germanium oxide (GeO,, 6.25 in 1000) induced in
several days dry necrosis of the tumoral lobe treated. I n less than a
month successive treatments induced the necrosis of the entire tumor.
There were no recurrences in three out of eleven cases. When the
recurrences appeared, after further treatment they necrosed. Molybdie acid (Moos, 9.25 in 1000) and also cerium borate (3.22 of CeO, in
IOOO), except f o r certain gradations, had analogous actions. Necrosis
was induced more rapidly than that occurring normally?by stannous
tartrate (1.9 of Sn in IOOO), by zirconium tartrate (6.9 of Zr in 1000)’
and by aluminum chloride (10 of ALO, in 1000). Only germanium was
effective when introduced into the general circulation of the plant.
342
A. J. RIKER AND T. 0. BERGE
CROWN-GALL
BACTERIA
AND ANIMAL
TUMORS
While work on the physiological interrelations between crown-gall
bacteria and their plant hosts offers many excellent possibilities, that
between these bacteria and animals is rather discouraging.
The isolation from cancers in animals (including man) of organisms
resembling Phytomolzas tumefaciew, and capable of inducing typical
crown galls in plants, has been reported by several workers. Blamenthal, Auler, and Meyer (1924) isolated a bacterium closely resembling
P. tumefaciens from a human mammary carcinoma. This organism
was said to induce galls in sunflowers two to three months after inoculation. Organisms differing from P. turnefaciens in certain biochemical characters but said t o be agglutinated by antitumefaciens serum
of rabbits, and inducing proliferations in plants, were isolated from
rat tumors ( 1 9 2 6 ~ )and from a human uterine carcinoma (19263) by
Fejgin, Epstein, and Funk. With the culture isolated from the case
of human carcinoma, a gall was induced on only one of seven plants
inoculated. The organisms recovered from the gall differed from the
type injected and were considered to be modified by passage through
the host plant. Amormino ( 1 9 2 8 ~cultured
)
an organism from a human
cancer capable of inducing large tumors on Agave. From cancers of
mice, Kauffmann ( 1 9 2 7 ~ )isolated organisms which induced galls on
sunflowers, comparable in size to those of controls inoculated with P.
tumefacielzs. Other similar strains failed to induce crown galls (19273,
1930). However, of the bacteria isolated from hundreds of strains of
mouse carcinoma and from mouse and rat sarcomas, Ikedo (1933)
found only 13 strains (1.9 per cent) which resembled the crown-gall
organism in their biochemical properties. Kauffmaiin (1928b), Robinson (1927), and Stapp (1927) considered organisms similar to P. tumefaciens, isolated from malignant tumors, as contaminators, of no importance from an etiological standpoint. It has been pointed out that
many such organisms may be found on the necrosing margins of malignant tumgrs. While chance contaminatioris may occur, the evidence
seems inadequate t o show that the crown-gall organism may appear in
cancerous tissue with significant frequency.
Studies of the pathogenicity of the crown-gall organism f o r various
warm-blooded animals have yielded conflicting results in the hands of
different investigators. Some positive results have been reported
(Blumeiithal, Auler, and Meyer, 1924 ; Blumenthal and Auler, 1925)
in mice, with the so-called P M strains isolated from mouse carcinoma.
Inoculation of the crown-gall organism in the ears of rabbits was
found by Amormino (1927) t o induce a distinct positive reaction. He
stated that while injections of P. tumefaciens failed t o result in 8x1
acute fatal infection in dogs, rabbits, rats, o r pigeons, local reactions
were obtained i n some cases. Reichert (1925, 1927a, 3, 1928) found
that the P M strain was closely related serologically to Phytomoqzas
tumefaciens. He suggested that a virus associated with the bacteria
might be the true causal agent of tumor production. Some degree sf
ATYPICAL AND PATHOLOGICAL MULTIPLICATION OF CELLS
343
immunity was reported in rats treated with dead cultures of the P M
strain, against Blumenthal's tumor ; but the best results were obtained
by using naturally attenuated tumor tissue and suspensions of fresh
normal tissues (liver, spleen and muscle). Smith (1925) was favorably
impressed by the work of Blumenthal and his associates, but confirmation and general acceptance are conspicuous by their absence. Teutschlaender and Kronenberger (1926) found completely negative results
iii mice, although such substances are kieselguhr, edema fluid, or ascites
fluid were added to the inoculum. Borghi and Luzeato (1929), who
cite other papers on this subject, used both crown-gall cultures and
P M strains and obtained negative results in rabbits and rats, even
though kieselguhr or extracts of crown galls were added to the bacterial
inoculum. The injection of P. tumefaciens, alone or mixed with India
ink, kieselguhr, etc., yielded only negative results in about 200 experiments on mice, rats, guinea-pigs, rabbits, chickens, or salamanders
(Kauffmann, 1928b). The senior writer and his associates, during
their fourteen years' studies of crown gall, while making puncture
inoculations into plants, have injected pathogenic crown-gall cultures
into their fingers scores of times. No proliferation of tissue ever
followed. Conversations with various other investigators have revealed similar experiences. From the evidence available it appears
that the pathogenicity of P. tumefaciens for warm-blooded animals has
not been demonstrated.
A strong bactericidal action against Phytoinonas tumefaciens of
fresh human serum from normal individuals a s well as from patients
suffering from carcinoma and various other diseases was demonstrated
by Kauffmann (1928a). A thick suspension of organisms in saline or
bouillon was allowed to fall on the surface of a n agar plate, and a drop
of fresh, active, undiluted serum immediately added. After twentyfour hours' incubation a t 37" C. only a trace of the bacteria remained.
Kauffmann showed that the bactericidal action was dependent on the
thermolabile complement, and was not possessed by certain sera. Carcinoma serum was most active against P. turnefaciens and in lesser
degree against certain other bacteria. While the bactericidal power
of horse serum was found to be about equal t o human serum against
the crown-gall organism, the effect was less marked in the case of other
animal sera, becoming progressively weaker in the sera of dog, guineapig, and mouse. The temperature employed was unfavorable for the
growth of the crown-gall bacteria, and raises the question concerning
what the result would be at about 28" C. However, these results present further evidence against any connection between crown-gall bacteria and human cancer.
The temperature relationships of the crown-gall organism alone
make it seem unlikely that Phytomorzas tumefaciems would be pathogenic f o r warm-blooded animals. Smith, Brown, and Townsend (1911)
found that 37" C. was above the limit for growth of the bacteria in
peptone beef bouillon or on peptone agar, and close to the limit f o r
growth in milk or potato media. Exposure on agar or in bouillon f o r
344
A. J. RIKER AND T. 0. BERGlE
seven days at 37" C. to 37.4" C. destroyed the organism. Pate1 (1929),
however, found that the crown-gall organism seemed to survive passage
through the blood stream of rabbits and the intestinal tract of mice and
guinea-pigs. Riker (1926), as previously reported, found that P.tumefaciens did not induce crown galls on tomato plants maintained at temperatures above 30" C. With these considerations in mind, it is not
surprising that inoculations with the crown-gall organism into warmblooded animals have failed.
Pathogenicity of Pkytomonas tumefaciens for cold-blooded animals
has also been studied. E. F. Smith (1916e) inoculated frogs, fish, and
lizards with cultures of the crown-gall organism but obtained nothing
corresponding to the striking tumors which developed on plants. I n
certain brook trout inoculated in the eye-socket, small nodular growths
appeared in the eye-muscles and elsewhere, but the fish died before it
could be determined whether these growths were true malignant processes. A sarcoma-like proliferation in fish (Carassius) was noticed by
Amormino (1928b) following inoculation with the crown-gall organism.
From fluid in the abdominal cavity he reisolated the organism which
induced a crown gall on Agave. It was agglutinated by rabbit serum
immunized against P. tumefaciens in dilutions of 1 to 50, 1 to 100, and
1to 200. A soft diffuse tumor which later became fibrous and delimited
was induced (1927) following inoculations of a turtle with an emulsion
of this organism. Kostritsky, Toumanoff and Metalnikov (1924) found
that when the larvae of Galleria mellonella kept at room temperature
were inoculated with P. tumefaciens, death resulted in each case. At
37" C., however, 9 out of 10 of the inoculated larvae recovered without
tumor formation. Thomas reported cellular proliferations and high
mortality in Nereis diversicolor (1931a, 1932), intense inflammatory
reactions and proliferation in Ascidia mentula (1931b), and death of
Sipunculus mudus (19314 following injection of P. tumefaciens. Certain other effects have been considered in the paragraph dealing with
mit ogenetic rays.
I n connection with work on animals it might be noted that crowngall bacteria form vitamin B in amounts effective in animal feeding
experiments, but apparently do not form noticeable amounts of vitamin
A (Burrows, 1925; Burrows and Jorstad, 1926).
The various aspects of the crown-gall problem reviewed in the preceding pages bring one to the question of how continued work with
plants may elucidate the cause of atypical and pathological multiplication of cells.
DISCUSSION
The complexity of the problems underlying the atypical and pathological development of crown-gall cells is apparent from the preceding
review. Certainly the basic researches have not yet developed sufficiently to permit a determination of the cause for the pathological cell
multiplication observed. However, it appears worth while to outline the more promising groups of working hypotheses suitable for
ATYPICAL AND PATHOLOGICAL MULTIPLICATION OF CELLS
345
further investigations, and to consider methods for determining their
validity.
The causal agent of crown gall is a bacterial organism. Of the other
hypertrophic and hyperplasial diseases of plants, some are caused by
different bacteria and some by certain physiological disturbances.
Knowing that bacteria may stimulate growth does not, however, furnish
an answer to the problems under consideration. The question immediately arises concerning what metabolic changes the bacteria induce to
cause the stimulation. Thus one arrives at a position similar to that
of workers who study the cause of growth not associated with bacteria,
except that the easily manipulated bacterial cultures provide an exceedingly useful tool in these basic investigations.
The study of metabolic processes of the bacteria, in association with
those of the host plant cells (see Fig. Z ) , forms one logical center of
attention.
This and other desirable approaches to the questions thus raised
are considered after mentioning certain ingenious concepts which seem
less promising. The latter either lack the background or present too
great technical difficulties t o place them among the more feasible lines
of attack on this problem. Until the experimental evidence or procedure in their favor is improved, it seems desirable to consider them
with reservation. Among these may be mentioned the concepts dealing
with mitogenetic radiation, filtrable material resulting from the action
of a bacteriophage, ultramicroscopic stage of the bacteria, and "tumefaciens-plastin. ' '
Some Promising Subjects for Further Study: The more promising
approaches to the question of cell stimulation present problems of great
complexity, often intricately interrelated or overlapping. For studying
possible cell-stimulating factors, certain considerations appear, such as :
(1)their nature, (2) their specificity, ( 3 ) their mode of action, and (4)
the continuity of their action. A short elaboration of these considerations and of their bearing on the stimulating factors has been made.
This may assist in clarifying the general problem, and in directing attention to the more promising lines for additional experiments, providing it does not obscure the interdependence of various factors, and
does not given an unwarranted suggestion of simplicity.
(1) The nature of the stimulating agent (or combination of agents)
may be chemical or physico-chemical. Among the chemical materials
produced by certain cell-stimulating bacteria may be mentioned : by
Phytomonas turnefaciens, carbon dioxide, ammonia, gum, various nonreducing carbon compounds and doubtless many other substances such
as bioses, etc., not yet determined (there are also reports of acetone,
alcohol, etc., mentioned earlier) ; by P. rhixogenes, in addition to these
substances, pyruvic and acetic acids. A long list of chemical substances
reported to cause cell stimulation has already been considered. Among
the physical-chemical changes induced may be listed disturbances in
oxygen supply, respiration, -SH and related regulatory equilibria,
oxidation, enzymatic activity, osmotic pressure, surface tension, cyto-
346
A. J. RIKER AND T. 0. BERGE
plasmic viscosity, cell-protein equilibria, and electrical equilibria including polarity. Practically any of these factors might induce local
irritation. The energy involved in the bacterial metabolism, which
produces these products and changes, seems to have received little or
no consideration. This energy employed in the intercellular spaces
seems well worthy of attention in relation to its effect on the surrounding cells.
(2) The specificity of the stimulating agent (or combination of
agents) needs clarification. It is possible that the agent is associated
only with the cell-stimulating organisms. It is also possible that the
agent is commonly produced by many microorganisms, but that among
them only the cell-stimulating forms are able to overcome the defenses
of the host cells and to live in metabolic association with these cells so
as to induce the stimulating factor. It may also be largely the result
of metabolism by the host cells. I n this connection perhaps the question of mutations induced in the host cells should be mentioned.
( 3 ) The mode of action of the stimulating agent (or combination
of agents) may be by supplying growth-encouraging materials or by
removing growth-inhibiting substances. Among such materials may be
mentioned several classes: ( a ) raw materials of growth such as ammonia, carbon dioxide, minerals, and simple organic forms of carbon
and of nitrogen ; ( b ) energy-producing materials, such as simple sources
of carbon ; ( c ) growth-promoting substances such as hormones, bioses,
auxines, carcinogenic agents, and earlier mentioned metabolic products
of the bacteria; ( d ) growth-inhibiting or controlling substances. It is
apparent that the removal or neutralization of a growth-inhibiting
substance might yield results resembling those from the addition of a
growth-stimulating substance. These growth-controlling materials are
important in the normal development of an organism. While many
questions involved in the mode of action are difficult to approach, many
others lend themselves well to experimental examination.
(4) The continuity of action seems important, since the agent which
initiates the pathological growth may be different from that which
maintains it. Consequently, if a causal factor were found for the early
stages of gall development one should also be sought for the later
stages. It has been suggested that the hyperplastic cells might provide
products capable of stimulating the rapid cell division. However this
may be, the evidence that the crown-gall bacteria disappear from an
actively growing crown gall appears defective. The possibility that
the bacteria and hyperplastic cells may supplement each other is worthy
of study.
Throughout these various considerations of causal factors, as has
been mentioned in earlier sections, it is important clearly to differentiate between cause and effect. For example, in the consideration of
differences between gall and healthy tissue, the conspicuous differences
in certain characters may be helpful in a study of causal relations, but
they seem more likely to be the result of a pathological condition than
to be connected directly with the stimulating factor.
ATYPICAL A N D PATHOLOGICAL M U L T I P L I C A T I O N O F C E L L S
347
Some Ezperimental Procedures f o r Examining Possible Causal
Agents ;The experimental examination of available material in relation
t o the suggested causal factors has been confronted with great obstacles.
While therc have been many working hypotheses for the cause of the
stimulation of atypical and pathological multiplication of cells, there
have been relatively inadequate experimental means for determining
their validity. Accordingly several recently developed methods, when
added t o Smith's (1917) earlier technic, provide useful procedures for
the investigator.
These experimental methods may be listed as follows :
(1) An extension of Smith's ( 1 9 1 7 ) method f o r the direct application t o the host of the factor or factors under consideration. When the
host tissue is properly treated with the agent being studied, a distinct
response in comparison with suitable controls may be secured.
(2) T h e application of these factors t o susceptible plant tissue growing in vitro. White (1933) has grown susceptible tissue in vitro for
many generations. It appears that certain types of experiments may
be performed much more easily on plant tissue in vitro than under
natural conditions.
However, inherent difficulties with both of these methods emphasize
the importance of certain relatively indirect approaches.
(3) Correlation of these factors with the comparative physiology of
various cell-stimulating bacteria. Pinckard (1935) has made parallel
studies on several organisms in this group. The characters in which
these bacteria are similar seem more promising f o r an association with
the factor causing cell stimulation than the characters in which they
differ.
(4) Correlation of these factors with the activity of both pathogenic
and non-pathogenic single-cell sister cultures (Hendrickson, Baldwin,
and Riker, 1934). The causal agent, if specific, should be present with
the pathogenic culture and absent from the non-pathogenic culture.
( 5 ) Correlation of suggested factors acith critical temperatures
(Riker, 1926). As mentioned earlier, inoculations on tomato plants
induced crown galls below 28-30" C., but not above this temperature,
although both host and parasite grew well in each case. Consequently,
the causal agent for tomato should be present at 27" C. or below and
absent at 31" C. o r above.
I n conclusion it may be said that further studies on the working
hypotheses briefly outlined, by means of the experimental procedures
suggested, seem t o carry definite possibilities for progress on fundamental questions concerned with the atypical and pathological multiplication of cells. Because (1) of basic similarities in the protoplasm
of living things, ( 2 ) of the common experience that advances in one
phase of biology aid progress in another, and (3) of the experimental
advantages provided by plant materials, it appears that in basic research on harmful growths the opportunities provided by plant cells
should be neither overlooked nor neglected. While it would, of course,
be rash to predict a solution of the problem, determination can be
348
A. J. RIKER AND T. 0. BEROE
made with the materials and technic available, which should either
verify or eliminate many working hypotheses on this subject. I n brief,
well directed studies with plant materials present excellent opportunities for elucidating many fundamental questions dealing with atypical
and pathological multiplication of cells.
SUMMARY
There are many different kinds of atypical and pathological cell
multiplication of living tissues resulting in such diseases as “galls, ”
“tumors,” o r “cancers” of both plants and animals (including man).
Although there are various working hypotheses concerning the cause
for such diseases, no satisfactory explanation has yet been given.
Without such an explanation efforts aimed at either their prevention
or their treatment are correspondingly handicapped. For basic researches in this field, plant materials have certain advantages over
animal materials : (1) large numbers are available at low cost, (2)
pathological growths, such as crown gall caused by Phytomonas tumefaciems, may be induced at will, and ( 3 ) both the pathogenic bacteria
and host plants are easily manipulated experimentally. Because of
these advantages, and because the basic protoplasm is fundamentally
similar in both plants and animals, some of the more pertinent literature on crown gall has been examined to clarify the possibilities, by
studying such a plant disease, of elucidating the general problem,
Abnormal and injurious tissue growth on plants occurred regularly
after the entrance of crown-gall bacteria through wounds. These organisms were found located primarily in the intercellular spaces, ruptured vessels, practically dead large exterior cells, and on the surface,
When the non-motile bacteria were introduced into a needle puncture
they probably moved between the cells because of capillarity or negative
pressure in the intercellular spaces. Motile forms migrated wherever
there was continuity of sufficient fluid.
Any living host-plant cells were found to divide under the stimulus
provided by crown-gall bacteria, but growing tissue was most active.
The cells immediately about the bacteria commonly divided first and
thus indicated the location of the organisms. Several weeks after
inoculation the crown gall was composed of irregularly intermingled
masses of hyperplastic, hypertrophic, and disorganized vascular tissue.
“Tumor strands” and “secondary tumors” occurred only following
inoculations at the top of plants having a number of internodes condensed in the apical buds. They were apparently not the results of
outgrowths from primary tumors through the regions of least resistance. The original inoculation of a condensed bud in some cases
introduced bacteria past a number of internodes. When these internodes elongated, the original inoculum thereby became distributed some
distance up the stem. After a suitable incubation period, the “secondary tumors” and “tumor ’strands” appeared. “Tumor strands”
were often, but not always, continuous. “Secondary tumors” did not
always repeat the type of tissue found in the primary tumor.
ATYPICAL AND PATHOLOGICAL MULTIPLICATION O F CELLS
349
A number of cytological phenomena have appeared to be the result
rather than the cause of the pathological condition.
Crown-gall bacteria utilized a great variety of substances as sources
of either carbon o r nitrogen. The large number of materials employed
suggested an extensive enzymatic equipment.
Metabolic products formed from glucose by the crown-gall bacteria
and determined quantitatively have included : cellular material, carbon
dioxide, bacterial gum, and non-reducing carbon-containing substances
secured as compounds with heavy metals ; and by hairy-root bacteria,
in addition to these substances, acetic and pyruvic acids. Various other
substances have been reported in media following the growth of crowngall bacteria. Free ammonia was given off from liquid carrot extract
media, but not from corresponding agar media. Vitamin B was found
in cultures of crown-gall bacteria.
Physical-chemical changes reported in cultures often varied with
different media. They include : changes in reaction, lower oxidationreduction potential, lower surface tension, lower ionic concentration,
and changed osmotic pressure. Probably all of these metabolic products or physical-chemical changes might act a s local irritants.
A large variety of chemical substances, including extracts of bacteria and of bacterial cultures, has been reported to induce cell stimulation when introduced into plants. Apparently none of the reactions,
perhaps because of technical difficulties, has been so great as those
induced by the bacteria.
The presence of crown gall on a plant has induced little if any
resistance to subsequent infection, although a suggestion of resistance
has sometimes appeared because of less vigorous growth.
Although crown-gall bacteria have induced antibodies in experimental animals, the evidence of a similar antibody production in plants
needs confirmation. The agglutination of the bacteria in plant extracts
was accomplished by acid and related factors.
The evidence appears defective which favors the presence of a
bacteriophage or virus as the cause for cell stimulation in crown gall.
I n relation t o dissociation, the crown-gall bacteria have appeared
like many other organisms. Extreme types of dissociation forms attributed to this organism, and credited with cell stimulation in both
plants and animals, appear without a basis of satisfactory evidence.
Strong x-ray and radium treatments have failed to injure crown-gall
bacteria. Hertxian rays and mitogenetic rays have been studied i n
relation to crown-gall developments with results that are questionable.
Radium and x-ray treatment of crown gall of sufficient intensity reduced
o r prevented growth.
The host plant has been found to produce galls well only when
growing satisfactorily.
The temperature range 38 to 30" c. was critical for gall development
on tomato. Inoculations induced crown gall below but not above this
range, although both host and parasite grew well at higher and lower
temperatures.
3 50
A. J. RIKER AND T. 0. BERGE
I n comparison with healthy tissue, crown galls had a lower sugar
content and were relatively alkaline. The galls had more positive
oxidation-reduction potential, greater titratable acidity, increased cytoplasmic viscosity, and more of oxidizing enzymes, tannin, proteinaceous substances, glutathione, and related -SH compounds.
The evidence in favor of crown-gall bacteria causing tumors in
warm-blooded animals appears defective. Better evidence appears for
the production of disease in cold-blooded animals and insects.
A number of working hypotheses on the cause of atypical and pathological multiplication of plant cells may be grouped together, respectively, in relation to their (1)nature, ( 2 ) mode of action, (3) specificity
and (4)continuity of action.
Technic is available f o r either verifying or eliminating many working hypotheses by certain experimental procedures including : (1) the
direct stimulation of tissue growing naturally, ( 2 ) direct stirnulation
of tissue growing iw vitro, (3) correlation of the factor under investigation with the physiology of various cell-stimulating agents, (4)correlation with the physiology of pathogenic and non-pathogenic sister.
cultures, and (5) correlation with that of pathogenic crown-gall bacteria
and tomato above 28 to 30” C.,. where galls do not develop, and below
this range, where galls grow vigorously.
It appears that studies with plant materials offer an excellent opportunity, f o r clarifying many fundamental questions relating to atypical and pathological multiplication of cells.
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