FORMATION OF RESISTANCE TO RUST IN WINTER WHEAT HYBRIDS

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• Sequence Number: 1
• Publication Date: May 1, 1953
• Content Type: REPORT

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-I I
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Voitchishina, 0. N.
Formirovanie ustoichivosti k zzhavchine
u gibridov ozimoi pshenitsy
([Formation of resistance to rust in
winter wheat hybrids].
(1)
Selektsiia i Semenovodstvo, vol. 20, no. 5,
pp: 31-33. May 1953. 61.9 Se5
(In Russian)
FORMATION OF RESISTANCE TO RUST IN
WINTER NFEAT HYBRIDS
Trans. 481
(In full)
ByI
A. Antik
Creation of rust resistant varieties of winter wheat is one of the basic con-
trol measures against this disease. Therefore the development of means furthering
the formation in hybrids of rust resistance and its subsequent retention in a
variety when the latter is used for production, are problems of great practical
importance.
1
The first results of our research were published in 1951 . In 1950 our
observations showed that rust resistance in winter wheat hybrids can be increased
drastically by providing plants during the growing period with additional feeding
outside the roots with salts of calcium, phosphorus and potassium; by culti-
vating hybrid plants when rust contamination is absent (this was achieved by
dusting the plants, with sulfur which is a fungicide in regard to rust); by
spraying the plants with a sodium chloride solution. ccording to data in the
literature NaC1 acts as a funf.:icide which kills rust spores; but besides that, the
presence of sodium stimulates intake and greater accumulation of potassium in
plants. And potassium plays en important role in.wheatts resistance to rust. The
1950 research was continued in the some direction in 1951 on 19 hybrids of the
North-Osetiia state selection station.
1. Inter-varietal _hybrids of free pollination 'with selected male parents):
Osetinskaia 3 ubi leinaia Osetin + Osetinskaia 4); Osetinskaia 3 x (Iubileinaia
a ----------------------.--_ -.------------------------------------------ -----------
1. Voitchishina, 0, N, Zhurnal "Selektsiia i semenovodstvott No. 10, 1951
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Table 1 (P-32)
Effect of additional feeding outside the roots on_rust resistance in winter
wheat hybrids ( in %).
Hybrids Variant of the test
Osetinskaia 3 x! 2-year addit. feeding outside
Intensive-Affected Yield from
ness of plants plots (in
affection relation?)
w. brown to control
rust
Eritrospermum I roots . . . . . . . .
0
0 ;
132, 0
15 Results of 1-yr addit. feeding
3,0
10,0
117,0
2-yr spraying with NaCl . . . .
0,2
0,1
116. ,0
Results of spraying with NaCl of,
1st year . . . . . . . . . . .
317
1210
108,0
2-year dusting with sulfur. . .
9,5
25. ,0
184,0
esults of sulfur dusting 1st yr,
9,7
30,0
145o O
Control . . . . . . . . . . .
43, ,0
65,0
10000
Zemka x (Iubil-1 2-year addit.feeding outside
einaia Osetin 4' roots . . . . . . . . . . . . .
43,6
50,0
167,0
Osoti aska:ia -3 r .r Results of 1-yr adcJit.feeding
45, 0
50, 0
103,0
h`uba kaia :133 2-year spraying with TTaCl . .
4210
55, 0
150, 0
k.Eritrospermum :Results of spraying with NaCl
G-569 of lst year. . . . .. . . . .
40,0
55,0
102)0
2-year dusting with sulfur . .
39,5
65j.0
98P O
Results of sulfur dusting 1st yr.
68,9
75,0
95,0
Control
70,0
100,0
100,0
Use of these methods, even for one year, considerably decreases tie
intensity of disease and the percentage of plants affected by rust in the
following generation of hybrids as compared with the control; it also increases
the yield of grain by 10-16%.
l'.rtificial inoculation with brown rust of hybrid seedlings of the following
generation demonstrated that additional external root feeding and spraying of
plants with a NaCl solution for two years increase the resistance of hybrids
to brown rust to a higher degree than when these methods are applied for one
year. (Table 2).
Phenological. observations indicated that plants of hybrids which were ex-
posed for two years to additional external root feeding and to spraying with a
sodium chloride solution, proceed 3-4 days faster through growing stages as
compared with the control. Seeds of hybrids which were exposed to a two-year
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additional external root feeding and to spraying with a NaCl solution increased
the germination energy as compared. with control. (Table 3).
Table 2 (p.33)
Results of artificial inoculation of hybrids with brown rust (in
2-year additional 2-year spraying with Control
feeding outside roots NaCl
Hybrids
?ri Q-{ ?rl
CO O -I- ?
G U
't
~ art
fU q--=
Q
U
M, O +
U
"
U;
Cy =.
CJ i
' &0
5 r~ O' O .
?rl 4a ?ri 4 a ?R!
:O 4-, ? Q i-)
U 0
C m C.)
4-3 M CH
F-i r, CU
"
W G
-H
4H ri
GIs
CJ
H? .~F Cd
,e G
E-4 H.
i s r
4-4 H
=-.P C 4-4
9 C) R
.
4
P-4
Zemka x (Iubiloi- 10-25
1;2
45,0
5-25
1;3
45,0
25-65
3;4
100,0
naia Osetin +
Osetinskaia 3..
Kubanskaia 133 .~
Eritrospermum
G-569
i
Osetinskaia 3 x 5-10
1;2
40, 0
5-10
1;2
150,0
10-25
;3;4
:100,0
(Osetinskaia 4 4
Iubileinaia Osetih
1 Kubanskaia 133
+ Eritrospormum
G-569
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Osetin -o Osetinskaia 4 Kubanskaia 133 + Eritrospermum G-569); Eritrospermum
G-19 x (Osetinskaia 4 4 Kubanskaia 133 . Eritrospermum G-569 + Zemka x
(Iubileinaia Osetin . Osetinskaia 3 7 Kubanskaia 133 + Eritrospeemum G-569).
2, Of limited-freeaolliriation: Iubileinaia Osetin x branching wheat;
Osetinskaia 3x branching wheat; Osetinskaia 3 x E,ritrospermum 15.
3, Inter-varietal crossings of freeol?ination: Iubileinaia Osetin of
free pollination; Zemka of free' pollination.
4,, Paired crossings 177: Iubileinaia Osetin x Zemka; Iubileinaia Osetin x
EritrospermumG-720; Iubileinaia Osetin x Kubanskaia 133.
5. Paired crossings: Zamka x Iubileinaia Osetin; .rdito x Zemka; Zamka x
Ardito; Iubileinaia Osetin x Voroshilovskaia; Voroshilovskaia x lubileinia
Osetin; Eritrospermum G-720 x Zemka; Zemka x Eritrosperr..n.um G-720,
For external root feeding of hybrids was used a solution of a combination of
salts; Ca (NO3)2 1 part v KH2PO4 1.5 part per 1 L. of watere For spraying
hybrids with sodium chloride was used a 2,0 solution.
The expenditure of salt solutions for each spraying of 10 square m. plots
was 3-3.5 L. The spraying was carried out during the following stages of plant
growth: sprouting, tillering, booting and heading.
The hybrids were dusted with sulfur 6 times during the growth period., The
first dusting took place after the ap-..)esrance of the first pustules of brown
rust on Krasnodarka, a variety susceptible to it. The sulfur expenditure for
each dusting was 100-120 g. per a 10 square m. plot. The test was conducted
in triplicate.
In 1951 the effect was studied of a two-year use of additional external
root feeding, of spraying (begin p. 32 with a NaC1 solution and of dusting
with sulfur. Besides that was being clarified the effectiveness of the
results of the use of the mentioned processes for the increase of resistance
among hybrids of the next generation.
Field observations of brown rust and records of yields according to variants
of the experiment, showed that hybrids of alnost all the combinations when fed
additionally outside the roots, sprayed with sodium chloride solution, and
dusted with ground sulfur decrease the affection with brown rust and increase
the yield (table 1)..
It is seen from the data of table 1, that the use of additional feeding
outside the roots with a combination of Ca (ITO~)2 A KH2POduring two years as
well as spraying with a IIaCi solution and dusting with sulfur, decreases the
damage from brown rust and lowers the percentage of rust-diseased plants.
The yield from a plot in some cases increases by 30-60% as compared with the
control.
c
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Table (,po33)
Trans. 481
Energy of germination of hybrid seeds.
Hybrids
Speeding up of growaing,stages
(in days)
Energy of
Osetinskaia 3 x (Iubileinaia
Osetin + Osetinskaia 4)......,. 5 4
Iubileinaia Osetin x spread-
ing wheat.......... 5 4
Zemka of free pollination.... 5 3
germination of
1 seeds (in % to
Booting Heading Flowering
control
3 117,0
3 106,0
3 112,0
The research of 1951 justifies the following conclusions.
1. Additional external root feeding of hybrid plants with a mixture of
Ca, P, and K salts and spraying with sodium chloride solution, increase their
resistance to brown rust,
2. The character of resistance is stable and is passed on to the
following generation of hybrids. Increase in rust resistance in hybrids
suggests that these methods form resistance to rust in the hybrid material
of winter wheat.
3. additional external root feeding and spraying with a NaCl solution
increase the yield capacity of winter wheat hybrids.
4. Use of these methods increases the germination energy. of seeds of
hybrid, plants.
All this permits us to speak of a future in the use of the mentioned.
procedures for selecting wheat in regard to resistance tq rust.
L.11-Union Scientific-Research
Institute of Plant Protection
Laboratory of Plant Immunity
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Ryzhkov, V. L., and Loidina, G. I.
Vzaimsdeistvie virusa mozaichnoi
bolezni tabaka s miozinom. i aktinom
(1)
(Interaction between the virus of the
Mosaic disease of tobacco and myosin and actin)
Dok. Akad. Nauk, vol. 92, no. 4, 1953, p. 851-853
511 P444A
(In Russian)
Trans& 482
(In full)
By:
A. Antik
There are data in the literature on intcracticn between the virus
of the mosaic disease cf tobacco (TIC') and protarnine, some protein enzymes
and serum prcteins (1-3). It is cbvicus from these data that the behaviwr
of TMV is no different from thrt cf other proteins and that between the
iso-electric points cf the TNV and. its interacting protein.fcrmation of
an insoluble complex can be observed most cf the time.
Muscle proteins differ considerably from prcteins the interaction of
which with TMV was studied before. i'. V. Engel'gard's end h. N. Liubimova's
discovery of fermentative activity cf nycsin (4) and. the peculiarity of inter-
actions between myosin and actin hakes the study cf intc.ractic.ns between these
prcteins and TIN especially. interesting.
We used in all the experiments a myosin scluticn of 0.5 M KC1, which
ccntcincd abcut 1.3% cf myosin. The-purified TI'MV preparation was used in
a 0.2% aqueous solution. In tests in which the effect cf intcraetion be-
tween myosin and TNV was determined per titer of T!,IV, the titer was determined
according to the number of lesions on the leaves of N. glutinosa, and as a
solution cf virus protein to which a KC1 scluticn was added instead of myosin
served as a control. The cencenti,ticn of this salt in the experimental and
ccntrcl solutions was, cf course, maintained strictly equal. The actin so-
lution was prepared in 0.1--M KC1 by dissolving 1 g. of actin in 20 ml. of
liquid. For polymerization of actin 0.001 M MgC12 was also added.
In the first experiment 0.5 ml of myosin were combined with 0.5 ml of
TMV and water was added gr^dually until a precipitate was formed. This
precipitate was obtained with the 0.178 M KC1 concentration. In the second
experiment 0.4 ml. of nycsin were combined with 0.1 ml. of TIvIV, after which
water was added and the sediment was fc.rned at 0.2 M KC1. Pure nyc. sin is
separated cut cf the scluticn only at 0.05 M KC1 (5). The result obtained
provided. a basis tc assume that myosin formed. a ccnplex with TMV. The pre-
cipitates formed were centrifuged out and then suspended in 0.5 ml of water.
In the sEdiment and the supernatant fluid, was c':c ternined the titer cf the
virus (Table 1). This determination of titer ecnf irm.ed cur hyp(-thesis that
part of the virus passed intr the sediment.
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(2) Trans, 482
C
An attempt was mode to obtain threads from the myosin ?r TIN
complex. Fcr this purpose a mixture cf solutions of the nenticned
proteins was blown into water through a Pasteur pipette. The fcrned
thread broke down rapidly. It was not possible tc obtain durable
threads similar to the actcmycsin Ines. Under the influence of the
adencsintriphcsphcrus acid (l_TP) the EN and myosin complex was con-
tracted (?) similar to the actcrsycsin.
Further on, experiments were conducted in order to find cut h- -w
actin affects the interaction between myc sin and TIN; for this purpc se
all three components were combined (begin p. 852) in equal but
in one test Ti'V was added to gyosin before the actin and in anc ther -
actin was added first. In these experiments actin F was used (pc ly-
merized actin). To 0.3 ml cf the mixture 0.7 ml of water was added
and thus a precipitate was formed which separated cut me the titer
of the virus was deter-.mined only in the supernatant fluid. Besides
these mixtures that cf 0.1 ml TIN + 0.1 ml of actin 4 0.8 ml of water
was used. It can be seen from table 2 that the pr,csencc of actin did
not hinder the depression of the virus titer. Furthermore; the actin
itself appeared. to be able to depress the virus ccnsiderobly.
Table 1 (p. 852) Table 2 (p. 852)
Interaction between TAU and }:iycsin
(the titer cf virus is expressed in
the number of lesicns on 10 half
leaves of _,:,_ lutinosr)
o 4-3 Frncticr. being Clu
~T-TTc f virus E, 0 CH
~., ?titr^ted
of
11
test ccntr4N
Interacticn between T?`:V and myosin
and actin (the titer (f? virus is ex-
pressed. by the amount of necroses on
10 half leaves of N. glutin: sa)
io?rl., ?!>, .
4D -P 4-D
80
213 37.5
1*csin t TMV
i
:..-
Fluid . . .
123
231 53.2
4 actin 40
101
39.6
Sediment
55
226 24.3
ijcsin,1 r:.-:ctin
Fluid. . . .
41
91 45.
TI AV 34
83
40.9
TIN . o.ctin 17
107
15.8
The problem of interaction between. actin and T?:V was studied more in
detail. It was established. that the ncn-pc.lymerized actin (actin G) de-
pres.-:,es the virus similarly tc actin F. Thus in c:ne test with actin we
obtained 52 necroses while there were 185 necroses in the control, i.e.
retention cf activity was 6.6%. Further tests were. conducted with actin.
-2-
Titer c f virus- W ` ~'
ar 0
CO '0 P-~ H
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When there was into motion between Ti V and. this prc tein in distilled
water (pH about 6.2), the activity of the virus in each of several
tests remained 19.6%, 20.0%, 22.1%, 23.9%. In crcJcr to clarify hcw
pH affects the depression of the virus by actin, experiments were
conducted. with a glycccal buffer; with pH 7 we found in various tests
the following retention of activity: 8.5%, 16.5%, 27.1%, while with
ph 4.6 - ccrrespc.ndiingly 37.1%, 25.4%, 62%. Thus the virus is depressed
by the actin most strongly when the media has , neutral reacticn, and
least strongly at p14.6. The latter pH is very close to the isc-
electric point of the actin and at this point a cloudiness of the sc-
luticn coul.0 be observed.
In c. rdcr to find cut how a h.Cl ecneentratic n affects the reacticn
which we are studying, an experir!ent was cc nductcc' with varicus salt
concentratic rs in the solution and while in 0.1 M K01 the retained ac-
tivity was 21.8%, at 0.25 M it was 49.3% -nd., finally, in 0.5 M - 80.5%.
Thus higher ccncentratic.n of KC1 leads to a decomposition cf the actin
and TIN cc.nplex.
Experiments were also cc nducted. (n the influence of 1 TP on the cc m-
plex. The results of these experiments ore shown in table 3 from which
it is seen thrt ATP itself depresses the activity of the virus negligibly,
but at the same time it decreases the effect cf the actin since it is ap-
parently capable cf breaking down its complex with the TIC.
Viscc sirnctric studies were also conducted and it appeared that the
combination if actin F with TIN does not produce an increase of viscosity.
. _s tc the mixture of myosin with TIN, the obtained result was not sufficient-
ly definite. In some tests we observed a quite considerable increase in vis-
ccsity, in ethers it was entirely lacking cr was very slight. In cases where
the viscosity was noted. it depended' greatly (begin p. 853) on the ratio be-
tween myosin and TMV and it reached a maximum when the ratio of mycsin to
TIN was 5 :4. In cne test with this ratio the speed cf running-off of the
fluid in the visccsi aeter was 2 min. 20 seconds while f-' -r the TIN it was
49.2 sec., fcr water - 44.2 sec. and for mycsin - 1 min. 44 sec.
C
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(4)
Trans. 482
Table 3 .(p.853)
Interaction between the TMV and actin and adenosintriphosphorus acid (the titer
of the virus is expressed by the amount of necroses on 10 half,leffives).
Combination
(mixture?)
Titer of virus! % of retained activity of
virus
a
Test Control
VTM A actin 23 115 20.0
VTM.+ 0.05% ATF .................. 57 77 74.0
VTM actin + 0.05% ATF ........... 26 79 32,0
VTPI actin ............... ........~ 79 334 23.9
VTM 0.0125% ATF ......... 112 11 1,10 62.2
VTM + actin T 0.0125% ATF.......... 121 326 36.9
VTM + 0.025;" ATF ................... 158 1 256 61.7
VTM + actin + 0.025% ATF........... ; 140 265 52.8
C.
C
The obtained results differ from what has been known before on inter-
action between T;'W and various proteins in that in our tests it was possible
to observe the formation of a complex at pH located above iso-electric points
of interacting components and not in the interval between these points. It
is known that the interaction between myosin and actin is observed also at
pH levels above the iso-electric points of those proteins, which is related
to their selective capacity to adsorb ions of potassium as well as other not
yet sufficiently studied properties. It is possible that due to varying
capacity to connect ions of potassium in interacting proteins a difference is
created in their charge which leads to the formation of complex (5). That the
interaction between TMV and actin, at least in part, is similar to the inter-
action between myosin anc? this protein is seen from the fact that the complexes
of TiW with actin depend on the concentration of KC1 and are broken down by
A`IP as are the complexes of myosin with actin.
?1e do not know yet the substrata in the plant's protoplasm with which the
THV connects, however it has been demonstrated that the development of a virus
infection can be suppressed by certain concentrations of potassium and mag-
nesiuri salts (6 & 7). It is possible that the TMV forms a complex with
structural proteins of the plant's protoplasm, which (complex) can be broken
down by high concentrations of electrolytes. However, the solving of this
problem requires further research ou plant proteins sensitive to TMV.
In conclusion the authors consider it their pleasant duty to thank
U.N. Liubimova for providing myosin and actin preparations as well as for her
valuable consultation.
Institute of Microbiology, AN SSSR Received: 1.VIII.1953
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(5)
Trans. 482
BIBLIOGRAPHY
4. Liubimova, M. N., Engel'gardt, V. A. Biokhimiia, 4, 716 (1939)
5. Sent-D'erd'i, A. On muscular activity. M. 1947.
6. Ryzhkov, V. L., Smirnova, V. A. DAN, 55, no. 3 (1947)
7. Ryzhkov, V. L., Sriirnova, V. A. ilikrobiologiia, 17, no. 3 (1948)
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Ryzhkov, V. L.
Elektronnaia mikroskopiia
dckletochnykh form zhizni
[Electron microscopy of pre-oell forms
of life]
Priroda, v. 4n, no. 9, pp. 48-52,
September 1951. 410 P933
(In Russian)
Trans. 483
(In full)
By:
A. Antik
ELECTRON MICROSCOPY OF PRE-CELL FORTIIS OF LIFE
O
C
The electron microscope made it possible to conduct direct observations
on objects which, due to their size, are beyond the range accessible to
light optics. It refers first of all to the minute biological individuals
the more existence of which we could judge before only by their activity.
Thus it became accessible to study the causing agents of the most severe
illnesses.
The first objects of electron-microscopical observations wore the
filterable viruses, but recently other minute agents of illnesses are being
studied as well as filterable saprophyte microbes which in their size are close
to viruses.
The data of electron microscopy indicate that the world of pre-cell life
forms is extremely diversified in sizes, forms and structures.
_ Rickettsia, representative of which is the causal agent of spotted typhus
L fever,, belongs to minute organisms. Unlike the viruses thoy disclose
respiration also outside the cells in which they parasitize. It was possible
to see through an electron microscope that the Rickettsia are surrounded
with a quite extensive mucous membrane. No other group of pathogenic agents
from the ultra-microbe world has a similar membrane.
Very peculiar are the '1tsistotsetylt L L-form]. To their number belong
the causal agents of pneumonia of cattle, of "agalaktiiaU of goats, as well
as agents of very little studied diseases which are manifest in malignant
rheumatism and other afflictions. To the same group belong also the saprophyte
forms which it was possible to isolate from sewage. The ""tsistotsety""
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(2) Trans. 483
Z L-forms] are very sensitive to streptomycin which cannot be said about
other groups being examined here. They look like very tender small bubbles
lacking any kind of wall. Sizes and forms of one and the same species of
"tsistotsot" vary, which indicates the complex development cycle through which
they pass. _
There are only single electron-microscopic data on "bartonelia" Bartonella .
They are causal agents of pernicious anemia and affect the red blood corpuscles.
In a regular microscope they look like minute rods, and with the help of an
electron microscope it was possible to disclose that these rods are not
individuals but represent aggregates of smaller single particles which, in size,
are close to viruses. The position of "bartonelia" in living Nature is very
unclear.
Among the viruses proper the largest are the representatives of the
psittacosis group (fig. 1). Psittacosis is a disease of parrots, its virus can
cause severe pneumonia in humans. To the same group belong viruses causing
pneumonia in cats, mice, and also the so-called "fourth venereal disease" in
humans (infectious limphogranulomatosis). Particles of these viruses are
apparently very rich in water, and since with the help of electron microscope
only dried out preparations can be studied, the particles of these viruses
have to be examined when they are quite out of shape which does not happen
to other viruses. Virus particles of the psittacosis groups vary considerable
in size and fora. The pneumonia virus of cats is 350-770 run in diameter.
As far as it can be judged according to electron-microscopic observations,
the large particles at certain development stages break up into smaller ones.
The viruses of the psittacosis group are similar to microbes in that they are
sensitive to sul.fanilamides, penicillin, aureomycetin and chloromycetin.
Viruses examined further on do not disclose any sensitiveness to either of
these substances.
Fig. 1 (p. 49). Piltcrabl4
viruses according to,data of-
electron microaaopyg
Pneumonia of cats
Pneumonia of mice
Psittacosis of parrots
Limphogranuloma
(fourth venereal disease)
Ornitosis of pigeons
1ieningo-pneumonia of mice
Atypical pneumonia of humans
Variola (srjall pox) of birds
it -t it of humans
Contagion mollusk of humans
"Ektromeliia" of mice
"Muksoma" of rabbits
Zostcr of humans
Chicken pox of humans
Herpes of humans
Mumps of humans
Pseudo bird pest
Bird pest
Grippe B of humans
Grippe A. of humans
Bacteriophage of intestinal
bacillus T2
Encephalomyelitis of horses
Bacteriophage of intestinal
bacillus T7
Papilloma of rabbits
Mosaic of beans, pumpkin, of
tillering of tomatoes
Poliomyelitis Lansing
mosaic of turnip
Grippe of pigs
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(3)
Fja. 1 (cont'd)
Mosaic of tobacco
X-virus of potato
Curliness of tobacco
Yellow jaundice of the silk worm
Granulosc of insects
Fig. 2 (P. 50)
Virus of small pox vaccine under electron microscope.
The preparation is dusted with metal which increases
the sharpness of picture. lagnified about 22
thousand tines.
Fib. 3 (p. 50)
Virus of grippe under electron microscope.
A quite homogeneous group is composed of viruses, as representatives
of which could be considered the causal agents of small pox. The elementary
corpuscles of these viruses have been long ago and in detail studied with
Q the help of light optics. Seen through a regular microscope they look like
minute spheres. Electron-microscopic observations showed that in reality
they are not globular but rectangular. (Fig. 2).
Very peculiar is the causal agent of the pseudo pest of birds. Placed
into a table salt solution it forms a long branch and. being transferred into
distilled water the virus particles become rounded. It is possible to
repeat the transition from one forr-., into the other several times, but if the
virus with the branch is killed with vapors of ns.c7 osmic (formic?) acid,
then when transferred to water it will not become rounded any ;.lore. Both
virus forms are infectious. In all these viruses the di-_''ncter of the particle
is above 100 m;-hin diameter. Another virus group has 100 ran (microns) and
less in diameter. The not studied representative of this group is the grippe
virus. Its various strains are 78-103 mm in diameter (Fig. 3),
The particle of the encephalomyelitis of horses is even smaller, it hardly
reaches 50-51 mn Zi_nicron?7. Of similar size is the virus which causes papi-
llona of rabbits, though in other respects it has no likeness with the causal
agent of encephalomyelitis,
large number of viruses which affect plants are 25-30 mn in diameter.
This size is inherent to viruses of -the southern mosaic of beans, of tillering
of tomatoes, of pumpkin mosaic and tobacco necrosis. Causal agents of turnips
and of streaking of beans are still sx.aller, they arc 17-19 mn 5icro7 in
diameter. To the viruses affecting animnals and humans belongs also the agent
of the hoof and mouth disea'e which is 20-30 mn in diameter. Similar sizes are
indicated for cone s'o cies of the poliorayelitis (viruses), however it is not
yet achieved to obtain these viruses, which affect animals and humans in form of
C
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(4) Trans. 483
sufficiently purified and concentrated preparations and the electron-
microscopic data pertaining to them is rather disputable?
Among viruses which have rod-shaped particles, deserving attention
are first of all the agents of the polyhedral diseases of insects, Called
polyhedra are the crystal-like corpuscles, which form in the gut, of
caterpillars sick with yellow. jaundice. It appears that these bodies
have a very complex structure,, Besides the albumen substance with particles
which are 10 mn (micron) in diameter and were first taken for the virus
itself, polyhedra have also virus particles which are quite ck rods.
The virus of the polyhedral disease of the mulberry silk worm is 88 mn
(micron) in diameter and 350 mn (micron) long.
Virus particles of the yellow dwarfiness.of potato are also rod-
shaped and their size is 200 x 50 nn (micron)?
Follows a large number of viruses which have the shape of thin
rods 1.3, (,macron) in diameter. The length of the rods is 150..300 nn
and these rods not infrequently join each other at the ends forming
threads which might reach several thousands of mn(micron). To viruses
having such structure belong the viruses of the mosaic disease of
tobacco, cucumbers, cabbage, potato, orchid, streakiness of peas (pea
streak), etc. Similar structure was for some time attributed to some
species of the poliomyelitis virus, but it has not been confirmed.
Thus the forms and sizes of viruses vary greatly. We have all
gradations of sizes beginning with the giant forms of the virus of cat
pneumonia which reaches the size of the smallest microbes and finishing
with such as the virus of the mouth and foot disease and mosaic of
turnips the particle volume of which is about 40,000.smaller than the
volume of an elementary corpuscle of largest viruses. Viruses vary not
only in form and size, but in their physiological peculiarities as well,
which becomes evident in their relation to antibiotics. Besides that
they differ also in their chemical composition, Viruses which contain
lipoids besides "neukleo proteidy" (?nuclear proteins?), are larger in
size than viruses lacking lipoids. The virus of the encephalomyelitis
and all the lar er viruses contain various lipoids, among them
phospholipids(?). In smaller viruses the presence of a small amount of
lipoids is known with certitude only in the virus papilloma. Absence
of lipoids in smaller viruses distinguish them essentially from protoplasts,
because each protoplasm, except albumen, contains lipoids. Bacteria
and higher organized beings contain two nueleinic acids--thymo-.
nucleinic and ribose(?) - nueleinic acids. The majority of viruses have
only one of these nucleinic acids, and, as a rule, the highest viruses,
i.e. the larger ones, contain the thymo-nueleinic acid. In the grippe
virus two nucleinic acids were discovered, in the virus of encephalomyelitis-
only the ribose(?) - nucleinic acid, same as in all the known to us
viruses affecting plants,, It can be said that a certain simplicity of
chemical composition o ? I f oxno
4ehydo
efttd
1:30
0 ? 30 noes
106 0 503 I.no
1s1200 ? M.2 min
Control Alive
r.
8 07 min o ? $9 mi.no ? 52 eec o
17 min. ? s o ? 10o7 man.
o Alive 3 hrs
2 Min o ? later ? 56 riin
Alive ? Alive r' Alive
o c
Pte- t 2n is an table I and 2 it Lo a raL tit tt r3alnti onn a3O c. J ~'s
the fuma o? b ?-=Ole alc n C3t$3r,;e, a 3-hm torU' n tii ,u E8f 1 'ekkm ?iv eb,.'.l?:`,', 1teo
which s er 1. s ob,jeetri in ouv Fri.=sires
T h e remits obtalmd justify the hypothesis t1mt lvnnole s ib which gams
in b y d r 6 ci9 of certmin gUmcceides m y 1 = ; a c c =
pa of t. plty ,oc.s;is9
cOU@1Ma
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tic tod ?
?
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1J
In the 110t, of such a ~ 'lac is O2 Vr.c wne the Trio cgie?a
of glueWIfts of the rsu's in ~dym which in 4i eaasa tf ' e- rot or
of wap r a tivvs of C rem may ,=4,, In of the eint ciibT ?
1k i b1W.Ggleallky r a .ffi thow s . bs es ,, vq n Injury of the =t
easlly lly fr two bio1og . - very s .vo aglueownt; zsi* aoicl
ss .=?ic a aria cop b e of parfam-Ing e as ter r&e in the SL,%vt: on
of rpe Sig wm*ips roaWk rOsftone wt,,
pf ective f um.4ion
Ay2wvnt1,ys it Is amok, only in b r e but P.- is otter rte, ret t that
of the Z "Alt's p is aslw in tub pc r j. l t ci+ n pro ;r w it "y
LUU,01 ((I:r aryl: a off igi .i8 .9 a ?i Q )a the eo t rn of
.uae , ic' s of t r~r ,iaa ?CUB& in the timn a of
them #
Of late,, there wammlated ftts ~ 5) vtriflaG theft at r E
gcis< lU ire Inve a shme f ; pro iww g of Otto Po s y
tbl@ cce3pii sg e of the Rion of a u m o s i c w t c1s1 cm1 raM in
the p ?t wr its s ? ti, untSM ; 'o"t't nWLy eaa*al.uz cl n e a1vr* ?
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(1) Trans. 488
(In full)
By:
A. Antik
p
Kamerat, A. Ia,
Novye ?itoftoreuctoichivye i
rakoustoichivye Sarta Kartofelia
New Phytophthora - and wart-resistant
varieties of potatoes].
Sad i Ogorod 1x45-48.
80 Sal'3
(In Russian)
New Phytophthora - and wart-resistant
varieties of potatoes
The September Plenum of the TSK YPSS (Central Committee of Commu-
nist Party of Soviet Union) set a 'goal - to bring, during the coming
two - three years, the production of potatoes and vegetables up to a
volume which would satisfy cortipletely not only the needs of the city
population and industry but also the potatoes needed by the livestock
industry. Of great significance in the complex of agro-measures
necessary for the completion of this most important task is the
correct selection of a variety and good quality of seed. The reso-
lution of the Council of Ministers of the USSR and of the Central
Committee of the KPSS "On measures for increase of production and
on store of potatoes and vegetables iii Kolkhozs and Sovkhozs during
1953-1955" obliges the Ministry of Agriculture of the USSR, Ministry
of Sovkhozs of the USSR, the soviet and agricultural organizations
to take measures for improvement of production of varietal potato
seeds, for a speeding up of reproduction and penetration into Kol-
khoz and Sovkhoz production of best-yielding varieties distributed
according to areas, particularly of early potatoes, in order to make,
by 1957, a complete switch to varietal potato plantings in all the
Kolkhozs and Sovkhozs.
Of particular importance is the adoption in potato production
of varieties resistant to the most dangerous diseases -- canker and
Phytophthora. At the present time our country possesses canker-
,resistant potato' varieties for various economic purposes and of
various ripening dates.
Up to the recent time in the world's potato assortment there
were no varieties resistant to the most dangerous disease - the Phy-
tophthora (late blight). It is not a quarantined disease; against
it the usual quarantine measures are worthless. The fungus causing
the disease appears everywhere where it encounters conditions favor-
able for its development. Almost everywhere in the basic areas of
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(2) Trans. 488
potato industry the moist and warm latter part of the summer creates
conditions favorable for intense spreading of Phytophthora. The
fungus affects the leaves and the tubers which causes a drastic de-
crease in potato yields. The sooner the Phytophthora appears the
grater can be the decrease in potato yields.
In some areas, for example in the Sakhalin oblast, the consider-
ably reduced yields of potatoes due to damage by Phytophthora has
a systematic character (regularly occurs).
Of quite particular importance is the disclosure in the vast
collection gathered by the All-Union Institute of Plant Industry,
of wild potato species resistant to Phytophthora.
Disclosure of such species made realistic the task of creating
full-value potato varieties more resistant to Phytophthora than all
the previously existing selections. For the first time in the world,
potato varieties with higher resistance to Phytophthora were created
in the Soviet Union. At the present time selection work for creation
of Phytophthora-resistant varieties on the basis of the initial mater-
ial from the collection of the All-Union Institute of Plant Industry
(VIR) is being developed in numerous scientific institutions of the
USSR.
Observations in various geographical areas indicate that Phyto-
phthora-resistant potato varieties which were not damaged in the
given area by Phytophthora through a number of years, in some years,
when the conditions were especially favorable for the fungus, showed
at the end of the growing period some degree of symptoms of Phytophthora
infection. This is due (begin P. 46) to a number of causes. There
is no doubt that the degree of aggressiveness of the fungus can change
depending on different conditions. In certain localities, under favor-
able conditions, the fungus can gradually become adjusted to the host
which was absolutely unsuitable before. Thus a more aggressive bio-
type of Phytophthora can be developed.
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(3) Trans. 488
This process of appearance of more aggr,ssive fungus forms is
furthered by the growing of potato collections which include diverse
interspecies hybrids having various degrees of resistance to Phyto-
phthora. .When Phytophthora appears early and the following condi-
tions in regard to temperature and humidity are favorable, the fungus
can transfer from less resistant forms (hybrids) to better resistant
ones, gradually acquiring greater resistance. Phytophthora acquires
the capacity to attack the more resistant hybrids at the end of the
growing season when changes due to age begin to take place in the
plant and make it less resistant.
Observations show that the more aggressive bio-types of the
fungus disappear the next year under conditions unfavorable for
Phytophthora development, particularly when spjcific control measures
are carried out against the disease.
In order to prevent the appearance of more aggressive bio-types
it is necessary in (variety) selecting institutions which maintain
potato collections to spray the plantings with Bordeaux mixture or
dust with the compound AB. It is necessary to select carefully and
to destroy the tubers which are to some degree infected with Phyto-
phthora and in no case should their planting be permitted. All the
potatoes set aside for sowing must be treated (chemically) in the
fall.
Under production conditions, two or more inter-species hybrids
originating from crossing with Phytophthora-resistant wild species
and having a varying degr..e of resistance - should not be grown on
one farm. Our observations in the Leningrad oblast show that in
growing one Phytophthora-resistant variety it is considerably easier
to avoid the appearance of a more aggressive bio-type of the fungus
even in years particularly favorable for the development of Phyto-
phthora.
It follows that it is not enough to create a. Phytophthora-resis-
tant variety, that a system of measures is necessary which would per-
mit retaining for many years the most valuable property-resistance
to Phytophthora.
In 1953, which was an exceptionally favorable year for the
development of Phytophthora; at the Belorussia state selection
station, the plantings of almost all the varieties, which were
resistant to this disease before, were damaged to some degree.
In the Leningrad oblast also some of the hybrids which in previous
years had no symptoms of the disease were partly diseased.
However, a great difference was observed in the Leningrad obla,at
between the incidence of Phytophthora in plantings of some hybrids
. , nd of regular varieties. In 1953, in certain areas of the oblast
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(4) Trans-. "488
C
Phytophthora appeared exceptionally early. The first disease symptoms
in the fields of early varieties (begin p. 47) were recorded in some
cases by July 5, and in others - in the'middle or in the third ten
days of July. By the middle of August on many farms Phytophthora
on many farms completely destroyed the leaves in the fields not only
of the early but also of the medium-late regular varieties distributed
for the oblast. By this time numerous Phytophthora-resistant hybrids
had no symptoms of disease. -By the end of August - beginning of
September the first symptoms of Phytophthora infection appeared on
less resistant hybrids. On many hybrids the first symptoms of Phyto-
phthora were disclosed by the middle or the end of September.
It follows that even in years most favorable for the develop-
ment of the fungus, the hybrids to soma degree resistant to Phyto-
phthora have an enormous advantage as compared with regular varieties.
If the disease even appears on them, it occurs considerably later
and, therefore, has no practical importance and harp y _n:.'iuences
the yield.
At the present time, in the All-Union Institute of Plant Indus-
try, a study is being conducted on methods for creating varieties
more resistant to aggressive bio-types of Phytophthora than the
C)
ones already existing. However, on the basis of data obtained under
varying conditions of the Leningrad and adjoining oblasts, the con-
clusion can be drawn that the varieties which are more Phytophthora-
resistant represent a considerable value and have to be introduced
into production as soon as possible.
The more widely distributed Phytophthora-resistant variety .in
the Leningrad oblast is the "Kameraz", selected by the All-Union
Institute of Plant Industry which is recommended for the Leningrad,
Pskov, Novgorod, Velikie-Luki, Kalinin, and Molotov oblasts. The
variety combines resistance to Phytophthora with that to canker
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(5) Trans. 488
(black wart). It is obtained by way of repeated crossings of the
wild Mexican variety Solanum demissum with the varieties: "Granat"
(twice), "Narodnyi", "Lichingen". The variety was grown from seed-
lings in 1937, was turned over in 1947 for the state varietal testing;
was for the first time distributed to areas in 1951.
The tubers are round, white with a flat top, slightly indented
"stolonnyi sled" (stem end). The skin is netted. The eyes are few,,
small, sometimes deeper at the top, not colored. The flesh is white,
does not darken when cut.
The corolla (petals) is medium large, white, sometimes light
violet on the outside. Formation of berries (fruit) is abundant.
Medium-ripening variety, for table consumption; productive. Tubers
of good taste,store well. As a shortcoming should be considered
lack of resistance to the fungal disease--macro-sporiosis. When
agrotechnique is inadequate and a systematic seed industry is lacking,
then sometimes symptoms of degeneration can be observed which are mani-
f>st mainly in the form of spottiness. Testing at the station r.sulted
in an average yield of 433 centners per 1 hectare from 1945 to 1950;
starch content on the average 14.5 - 15.0%.
In tests in state varietal plots this variety usually occupied
one of the top plac:s in regard to yield capacity, producing, for
example, in the Leningrad varietal plot 109 - 133%, in the Luzhsk -
108 - 120%, in the Gatchina plot - 114-125% in ratio to the standard
variety "Barlichingen". The starch of the "Kameraz" variety is always
higher than that of "Berlichingen". Thus in the Leningrad (b-,gin p.48)
C
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(6) Trans. 488
varietal plot the mean starch content of tubers of the "Kameraz"
variety during 1947 - 1950 was 14.72%, almost 1% higher than that
of "Berlichingen". In regard to the supply of. starch on the average
for 1948-1950 the potatoes of the "Kameraz" variety presented 123%
of the standard and according to the mean weight-of the commercial
(?) tubers - 132%.
Especially significant was the behavior of the variety in 1953
in the Leningrad oblast when the Phytophthora was spread intensely.
At the experimental base of the VIR "Krasnyi Parkhar"" near
Leningrad in production fields when square-hill planting of the
standard variety "Berlichingen" produced a yield of 160 centners
per 1 hectare the "Kameras" variety produced 270 c. per 1 h. In
the Kolkhoz imeni Lenin, Gatchina raion, the potato yield of the
"Kameraz" variety in a 48 hectare area was 160 to 350 c. per 1 h.,
in the Kolkhoz imeni Ihdanov, Volosovskii raion, - 185 c. per 1 h.
(and "Berlichingen" variety 117 centners), in the sovkhoz "Krasnaia
zaria" in large: production areas the yield reached 300 c. per 1 h.
A considerable surpassing of yield capacity in comparison with
varieties non-resistant to Phytophthora is recorded also in other
Kolkhozs and Sovkhozs of the Leningrad and adjoining oblasts.
According to data of the Sakhalin branch of the AN (Academy
of Science) SSSR, the mean yields for 3 years in the Sakhalin oblast
of the variety most widely distributed here "Mestnyialyi" -- was
200 centners per 1 hectE;r and of the variety "Kameraz" - 330 c.
Total wast_ of varieties was: "Berlichingen" - 15%, local - 6.6%
and of the:"Kameraz" - 1%.
In the same oblast, according to the material of the Aleksan-
drovskii state varietal plot, of the fifteen varieties being tasted
in 1952, the "Kameraz" variety stood out in its yield which was 327 c.
per 1 h.
Besides the medium - late Phytophthora - and canker-resistant
varieties, n.:eded for the Leningrad and the adjoining oblasts are
also productive early and medium early canker-resistant varieties
more resistant to Phytophthora than the already existing varieties.
The standard early variety "Cobbler" is to a high degree damaged
by Phytophthora which hinders its expansion. Other early varieties
are also seriously damaged by Phytophthora.
At the present time in a series of Kolkhozs and Sovkhozs good
results were obtained from tests of new varieties grown by the All-
Union Institute of Plant Industry: Pushkinskii (U8-236) and Detsko-
sel'skii (R8-168). The first is an early variety, the second --
medium-early. According to preliminary data both varieties are cankdr-
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(7) Trans. 488
resistant (the test is not yet completed) and more resistant to Phyto-
phthora (especially the Pushkinskii vari:ty) than the alriady existing
early and medium-early varieties.
Penetration into production of varieties more resistant to Phyto-
phthora is of tremendous importance for the increase in potato yields
.in the zone of extensive incidence of this disease. We have at the
present time varieties more resistant to Phytophthora and ripening
at different dates. It is necessary to reproduce the seeding material
rapidly and to test the varieties mentioned. At the same, time system-
atic seed-growing is a must, in order to sustain, on a high level, the
seed properties of new varieties. It should also be noted, that these
varieties produce good results under conditions of sufficient fertiliza-
tion and moisture (without excess water). Therefore, in cultivating
the Phytophthora-resistant varieties it is obligatory to apply the entire
complex of methods of progressive agrotechnique.
pansive entrance into production of more Phytophthora-resistant
potato varieties together with many other measures will further a most
rapid transition into life of the historical resolution of the Septem-
ber Plenum of TSK KPSS in regard to the field of potato industry.
9
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(1)
Zadina, J.
Die biotypen der karrtoffe1kraut-
f9ule (Phytophthora infestans) and
die resistenzzUchtung gegen die
kartoffelkrautf&ule.
Ceskoslov. Akad. Zemedel. Sborn.
261569-574. Dec. 1953.
19.5 C332C
German Summary
(In Czeck)
Trans. 489
(Summary)
Byt .
A. Antik
15 variants of phytophthora isolated in various locations of
Czechoslovakia are discussed in this article. By way of applying
these local variants to an assortment of 4 potato varieties (Aquilla,
Falke, Fridolin, Roswitha) which were resistant to phyto.phtora on tubers,
they were divided into 6 bio-types. The aggressiveness of these bio-
types was compared with that of the Polish bio-type which affects tubers
of all the selected potato varieties resistant to phytophthora. So far
this aggressive bio-type was not found in Czechoslovakia's territory.
Further on the article discusses the problem of originating of new
bio-types in the territory of the Czechoslovakian Republic (CSR) and of
the possibility of using wild potato varieties in breeding varieties
resistant to highly aggressive phytophthora bio-types. According to
tests carried out up to now, the Polish bio-type affects 60% of hybrids
of the wild variety Sol. demissum and cultivated varieties, while the
bio-type from Velke Karlovice affects only 20% of these hybrids.
489
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(1)
Afanas'ev, P. V.
0 mekhanizme i kinetike
fermentativnogo sinteza
[The mechanism and kinetics of
fermentative synthesis].
Biokhimiia .14(5)424-431.
Sept./Oct. 1949. 385 B523
Trans. 4.90
(In full)
By:
A. Antik
(In Russian)
C
Enzymes possessing only a Synthetic action are so far not known,
therefore it should be assumed that the same biological catalyzers pro-
duce reactions of decomposition as well as of synthesis. This corres-
ponds with requirements of thermodynamics, because in an opposite case
the enzymes could disturb the chemical equilibrium.. Tendency of the
process towards decomposition or synthesis depends only on the question-
in what case will the process lead to a decrease of free energy of the
system.
According to Oparin's (1) presentations the tendency of enzymatic
processes in the living cell is determined by adsorptive factors. In
some combinations the adsorbtive factors can oreate conditions under
which synthetic processes can proceed with a decrease in free energy
of the system, though individual components change with an increase of
free energy. A detailed presentation of the mechanism and a concrete
notion of conditions under which such processes take place in the liv-
ing cell, cannot be riven yet because of extremely limited information
? and an exceptionally complex system.
Simple cases of conversion (?) of enzymatic decomposition were
realized by many researchers. These cases have the peculiarity that
they take place with relatively negligible changes of free energy;
these changes can be compensated easily (and even change their symbol
(?sign?) ) with the help of experimentally accessible changes of con-
centrations of reacting components. In a case of high levels of
changes of free energy it is not possible to realize the conversion
with simple means. And the majority of the most important biochemical
processes belong to the latter type.
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(2) Trans. 490
One of the methods of conversion of the enzymatic hydrolysis -
which is biologically one of the most important ones - is experi-
mentally realized and theoretically substantiated conclusively by
Bresler and his co-~raorkers (2), who demonstrated that in a case when
the enzymatic process takes place with an increase in the volume of
the system, it is possible, with the help of external pressure, not
only to compensate but also to convert the symbol of change of the
free energy of the process. This permitted conversion of the enzymatic
hydrolysis of polypeptides and polysaccharides, i.e. to realize syn-
thetic processes. Theoretical deliberations and experimental results
obtained by Bresler are of great significance in principle for com-
prehension of biochemical processes and they open a wide perspective
for experimental and theoretical study of enzymatic reactions.
The purpose of the present work is to clarify the mechanism of
action of enzymes in a synthetic process, to make theoretical con-,
clusions of kinetic relations and to test experimentally these con-
clusions.
Our research was conducted on the example of hydrolysis with try-
psin of polypeptides under atmospheric pressure and re-synthesis of
hydrolysates under high pressure.
In the preceding report (3) - on the basis of notions, which we
developed, on the nature of enzymatic activity - an equation was given
of the relation between the speed of enzymatic hydrolysis of polypep
tides and the depth of hydrolysis
V o K3 FS - KlLK2S2 , (1)
K31 K1,#.2S
where V - is the speed of the process; F - concentration of the en-
zyme, S - concentration of the substrate (depth of hydrolysis); K1
K2 and K - constants of speeds of intermediate reactions. We demon-
strated that kinetic peculiarities of enzymatic hydrolysis of poly-
peptides are well described by the su,gested equation. This equation
(1) was derived on the basis of the hypothesis that enzymatic hydrolysis
proceeds according to the scheme
1
F. S
FS;
.
2.
FS ; F
K 2..`f
F2S;
3.
K
F2S .__
2F -2S-,
where F is - concentration of the enzyme; S - concentration of the sub-
strate; K1K2K3 - constants of speeds of intermediate reactions. The
active link which produces the reaction is the complex F2S. In this
complex takes place the activation of~the substrate which eases its
hrdrolyt:ic decomposition. It follows from equation (1), that the
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(3)
hydrolytic process has to proceed with increasing inhibition by the
substrate and the reacting system gets finally into a kinetic dead-
end., In this dead-end the reaction cannot take place. 'Due to the
fact that this dead-end is reached long before a complete break-
down of the polypeptide, it becomes understandable, why the experi-
mental depth of the ultimate hydrolysis is never great. On the basis
of equation (1) the condition of the kinetic dead-end is expressed by
the relation:
V= 0, when F=42_ S
In a case when the system, for example trypsin -- polypeptide, is
under conditions when thermo-dynamically advantageous is the process of
synthesis of peptide relations and not the hydrolysis---the enzyme will
catalyze the synthetic process to a higher degree. It is impossible
to suppose that in reversing the process of hydrolysis, all the inter-
mediate stages are fully and completely reversed. For that. we would
have to assume that the synthesis is realized in the act:
FS- F2S2 --a F2S
But this is impossible because the complex FS2 is inactive and there-
fore activation of the substrate cannot take place there. In previous
reports we expressed considerations about the reason why the complex
FS* is inactive. Therefore the intermediate stage, where the act of
synthesis of peptide linkage takes place, should be a stage which in-
cludes the active complex F25--. In this active complex takes place
an activation of the substrate which makes it capable of reacting with
water in case of hydrolyses or with another molecule of the substrate--
in case of synthesis. Therefore it is necessary to assume that the act
of synthesis of peptide linkage is realized in the following inter-
mediate reaction:
F2S* 4- S2 ---:) F2S
Accepting this hypothesis the scheme of ferment action with a synthetic
process can be presented in the following manner:
1.
F+S2_.. K FS-
2
K., F
S
FS-i-F
.
2
,
3.
F2S +S-'--.K3--; 2F+-S
l+ Since during a hydrolytic dissociation of the substrate (poly-
peptide) products are generated which are also a substrate, the
actual concentration of the substrate, as a result, increases.
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(4.) ' Trans. 490
Applying the principle of quasi-stationary property of centrations of
of intermediate products and expressing them through concentrations of
enzyme and substrate, we shall obtain an equation for the speed of re-
action:
K
V - dS K
_ ..._,1(FS - _._1 2
_- S )
dt Y l12 K 3 K2 (2)
Obtained.are a mechanism and equation of kinetics identical with
those which we proposed in the preceding report for catalase.
It follows from equation (2) that- K
V = 0 either when S 0 or when F- -?----1----S 0; i.e. when F....:11-5 or
K2 K2 K2
So (not clear) = Kl" F?
Differentiating equation (2) we shall obtain:
00
Kl . (F._2 KlS),
]. 1.2 ;K K2
K3
(3)
from where dv.___-.._ 0 when F .-2 ... Kl S - 09 i.e.
s K2
F = 2 _ or [ =K 2_._._
K2
1
Therefore the relation between the speed of reaction and the con-
centration of substrate, equation (2), has a maximum t 1:-.7 , i.e-.
the function is symmetrical ( maximum concentration of substrate
at which the speed of reaction is completely suppressed).
It is necessary to point out that equation (1) has an unsym-
metrical aspect and the maximum speed of rea.ct:i.on is realized at rela-
tively small, substrate concentrations.
From equations (1) and (2) follows that kinetics curves (S, T,)
where S is the substrate concentration and T---the(time) for hydrolysis
and synthesis have to be quite different in form. Under the usually
applied conditions for carrying out of enzymatic hydrolysis of poly-
peptides, the curve (S,T) is close in form to the normal type, i.:.,
at first the reaction speed is a maximum one and then it steadily
drops to zero. The kinetics curve (S,T) of the synthesis has to
have an unusual S - like (symmetrical) aspect.
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(5) Trans. 490
Experimental Part
In order to test the exposed theoretical deliberations and con-
clusions, we conducted following experiments.
Kinetics of hydrolysis with trypsin of egg slbumin (crystallized)
was measured: 1% egg albumin in borate buffer (0.2M) at pH 9.15 (be-
fore experimenting the albumin was denatured by heating in a boiling
water bath for 10 min.), trypsin 1/25 part of the albumin weight at
380. Antiseptic-thymol. Determination of amine nitrogen was con-
ducted according to the Van-Styke method. The results of measur-
ings of hydrolysis are given in fig. 1.
N mg/ml N mg/ml
CII
(P - 426)
Fem. 1.Hydrolysis of 1% egg Fig_2.(p.426) Re synthesis of
albumin in the borate buffer hydrolysate of egg albumin un-
(0.2M); pH 9.15 with trypsin der the pressure of 6,000 atmos-
being 1/25 part of albumin pheres at a 38?temperature.
weight. Temperature 38?,
antiseptic-thymol.
0
For a resynthesis under a 6,000 atmosphere pressure at 38 was
taken hydrolysate 22 hours after the beginning of hydrolysis and then
several samples of hydrolysate were placed under pressure for varying
time periods. At the end of the required period the ^pparatus was
cooled to room temperature, the pressure was removed and the samples
were immediately analysed by-the Van-Slyke method`-for content of amine
*.itrogen. The technique for conducting the experiments under pressure
is analogous to that described in Bresler's works. The results of the
experiments on synthesis under pressure are presented graphically in fig. 2.
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(6) Trans. 490
C
In comparing fig. 1 and 2 it is :apparent that the character
of .kinetic curves (S,T) corresponds completely with those expected.
It is necessary to point out that the kinetics of resynthesis ex-
perimentally studied by Bresler and Glikina on the system gf-latin of
trypsin, is not fully known. Our detailed. measuring of kinetics
of resynthesis on the system of egg albumin---trypsin is in complete
accordance with observations by Bresler and Glikina.
In differentiating the curves (fig. 1 and 2), are obtained curves
(V,S) of depe_.~ndence of speed on substrate concentration which correspond
to equations (1) and (2); they are presented in fig..3 and 4. It can
be seen that the curve (V. S) of fig. 3 for hydrolysis is indeed unsym-
metrical. The curve (V,S) of fig.?4 for synthesis presents a sym-
metrical curve. These results correspond fully with the requirements
of the above-dev(loped theory. Equation (3) requires a direct-line
relation between ---(IV and S.
In fig. 5 are presented the results of differentiation of the
experimental curve in corresponding coordinate axes. The direct-line
character of relation of experimental values coincides completely
with the requirements of the theory.
N mg/m1. hour
(p.42'?)
F j. Relation between the Fi ;,_4~ Relation between the, speed
speed of enzymatic hydrolysis of enzymatic synthesis under
end the depth of hydrolysis pressure and the depth of hy-
(concentration of substrate). drolysis (concentration of sub-
strate)
C
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(7) Trans. 490
0
0
FiL__5. Relation bet .r-. n derived sped of enzymatic synthesis
of substrate conce:entrati-on. rind thE. cone ntrat.i on of substrate.
Br,: sler and Gliki.n 6. Bu thman, M. P. and Manoilov, S. E. DAN 69,44, 1444
C
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(1) Trans. 493
(In full)
By A. Antik
Ermolaev, M. F.
Iz menchivost' form sushchestvovaniia
mikrobov i virusov nasekomykh
[Variability of forms of existence of
iaicr'obes and viruses of insects. ]
Agrobiologiia 4A89-99
Ref. July/Aug. 1953. 20 Ag822
Observations on Phytometra gamma
(In Russian)
VARIABILITY OF FORMS OF EXISTENCE OF MICROBES
AND VIRUSES OF INSECTS
iiore than 60 years ago the Russian scientist D. I. Ivanovskii (3)
called attention to the filterable and infectious properites and the
corpuscular nature of viruses, to their ability to be deposited in form of
crystals in tissues of plant organisms. The discovery of filterable
viruses enriched the world sciende.. The science obtained new facts on
the origin and development of life. This was a discovery in nature of a.
new, not previously known, form of existence of albumin bodies which h^ve
no cell form, but possess all the properties of a living organism.
P.fter P. I. Ivanovskii's discovery many diseases were disclosed in
the plant and animal world which are caused kyy :filterable viruses. At
the same ti._:e it was noticed that there is some connection between viruses
and known to us cellular forms of microbes. Thus, for exa.i:iple, Rickettsea--
causal agents of typhus--form elementary corpuscles; they are larger than
viruses, but considerably smaller than bacteria. Part of the microbes
appeared to be able to change to a visual form, which possesses the
property of producing a new visual cellular form of microbe.
But in spite of the -accumulation of factual material which allows to
record still new virus disuses of plants and animals, the nature of
viruses, their origin remained a puzzle.
On the basis of the metaphysical dogma that "a cell is only from a cell.",
some scientists attempted to explain the origin of viruses by a staple
breaking up of microbe cells. Others maintained that a virus is a dead
substance which reproduces in cells of a living orgainism auto-catalyt-
ically, i.e. by way of a mechanical change-over of -living albumin molecules
._g9-
X93
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l4J grans. Sao
(I
of an organism cell into a substance similar to it. The metaphysical
character of these hypothesis is obvious. Nevertheless experimental
proofs were required that viruses are not a result of a simple degradation
of cellular forms of microbes or their breaking-up to albumin molecule,
that viruses are not only a complex chemical substance which is outside
the development process, but a living substance as well, from which
during the development process more complex organisms can originate--
microbe cells, and that the latter can generate a structurally simpler
living substance--the virus.
A. considerable step forward in solving the problem of origin of
viruses were the works by 0. L. Lepeshinskaia. In studying the non-
cellular form of life, 0. B. Lepeshinskaia. (5) proved experimentally the
possibility of origin of cells from a non-cullular living substance and
thus rbfuted the idealistical and metaphysical notions of life which
follow from Virchowts reactionary teaching.
-89-
(begin p. 90)
ConetU>Ca.ble progress was achieved by V. A. Krestovnikova (9),
ED L. I. 'al'kovich, V. V. Suknev, G. M. Bosh'ia.n (1) and other researchers
who studied invisible forms of microbes of non-cellular structure and
the possibilities of their c.h-,nging (conversion) into visible cellular
forms.
Our experimental work with pathogenic microbes of harmful insects
which started in 1947 in the All-Union Scientific-Research Institute for
Flax, demonstrated that micro-organisms are able to generate, in the
process of their development, a non-cellular form of living substance
which preserves the capacity of reversibility of the process.
The initial object of our work were caterpillars of the cutworm
moths--ga , which are dangerous pests of flax and of many other agri-
cultural crops.
Observations of the cutworm moth-gamma development in 1935 and then
in 1947 established, that a sharp change in temperature and frequent
? precipitation further epizootics which leads to a mass destruction of
caterpillars, cocoons, butterflies and eggs of the cutworm moth-galinna.
The causal agent of the disease oLL tLis cat-,r?p~__-1-z
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CD
unknown until recent times, though there were indications (10) that the
disease is caused by micro-organisms and that a mass dying of caterpillars
is possible from polyhedral virus disease--yellow jandice.
In 1947 we isolated from organisms of diseased and dead caterpillars,
cocoons, butterflies of cutworm moth--gamma. a bactezium which differed
in its cultural and biological properties from previously described
pathogenic microbes causing disease of insects.
We called this microbe temporarily B act. Gamma Sp. nov_., which
corresponds with the species name of the insect organism from which it is
isolated.
In a 24 hour bacterial culture, the bacterial cells of Bact. Gamma
sp. nov. have the aspect of rods up to 2.3 microns in length and 0.55
microns in width, which are distributed singly, in pairs and rarely
in chains. The bacterium does not form spores, it is Gram-negative.
Changed (coccus-like and coccus-like with a transparent aureole)
forms of cells start appearing on the third-fifth day after sowing of
bacteria in nutrient media.
Bacterinlcnlonies in meat-peptone agar slimy at first transparent,
then cloudy-white. Bacterial cells developing in raert-peptone agar cause
a surface dissolving of agar in connection with which a large amount
of fluid is formed. Spreading on the surface of agar it furthers the
formation of bacterial film which covers the surface.
fig. ._I (p.90) Crystal-like cor- Fig. 2 (p.91) Crystals generated
puscles of yellow jaundice virus, in the bacterial film of Fact.
which originated in the organism Ganuria sp. nov. in the liquid
of a caterpillar inoculated with nutrient media
the microbe of Bacterium Gamma
sp nov.
The bacterium which we isolated decomposes (with generating of acid)
levulose, saccha.rose, galactose, mannite, arabinose.
(begin p. 91)
The Bacterium develops intensely in solutions of saccharose, levulose
and maltose. It reduces nitrates, coagulates milk and dilutes Olatin,
but it breaks down starch extremly slowly. Therefore sowing of bacteria
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t4) Trans. 493
m
in pot'.to agar produced a rather weak growth of colonies which stopped
completely on the third-fifth day after the sowing.
Bacterial colonies grow only with a free access of air. Deep inside
a solid nutrient media they did not form.
In a meat-peptone broth the growth of bacteria takes place also
onthe surface and then an uninterrupted thin dry film is formed which
covers the surface. The cloudiness of the both is moderate, constant,
the residue is porous during the first 24 hours, after that---flaky.
When the culture is kept long it (the broth) becomes thick and viscous.
Fluorescence of nutrient media was not observed. Optimum temperature
for growth is 15 - 17?, maximum--30 - 35? and minimum--3 - 50.
Testing of pathogenic properties of Bact. Gamma sp. nov. indicated
that artificial inoculation of c?terpillars of cutworm moth-gamma through
food with pure bacterial culture causes iass disease and destruction of
84 -.100% (Table 1).
Effect of Artificial Inoculation of Cdterpillars and Cocoons of
of Cutworm Moth-gamma with Bact. :arnma. s p. nov. (laboratory
experiment 1948')
Variant of experiment VTumber Number of
of cater- destroyed
pillars in~ in
I
the exper- catteer cocoon
iment pilla
phase
Inoculation of cater--
pillars with bact=
.eria through food
. 30
Inoculation of cater-
pillars of cutworm
moth,-mma ley Way..
of spraying the
caterpillar with
bacterial suspen-
sion.
Control (without
inoculation.
0
I
otal !Total of
of destroyed
c .n Jcaterpillars
butter-j& cocoons
phase flies Number
20 1 4
26
X00,10
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(5) Trans. 493
The most susceptible to inoculation appeared to be the caterpillars
of the third and fourth generations. Inoculation of caterpillars of the
fifth generation frequently caused their destructioh during the pre-
cocoon stage or during the phases of cocoon and butterfly.
_91-
(begin p. 92)
The butterflies which we bred from inoculated caterpillars, as a rule,
were not able to deposit eggs and if they did it, then in very negli-
gible amounts (not more than 20 - 30). Eggs deposited by diseased
females produced no breed due to their inner decomposition by bacteria.
Tests demonstrated that the bacteria culture which we isolated is
able to cause disease and destruction also of caterpillars of cabbage
butterfly, cabbage cutworm moth, clover cutworm moth, nettle rash,
potato cutworm moth.
Observing the pathological process in the organism of caterpillars
we noticed, that with the decomposition o ' inner organs of caterpillars
the microbe itself changes shhrply. At the. beginning of manifestation of
CD disease symptoms of caterpillars we disclosed in their cavity-fluid
microbe cells of rod-shape and then, when the decomposition of cells of
hemolymph and fat-albumin body began, the bacterial cells became very
small and in the cavity fluid appeared coccus-shaped cells with a
transparent aureole. Further on the picture charged again and at the
moment of complete deco::.position of the inner or_a.ns of caterpillars
their cavity-fluid was filled with a mass of clear rounded corpuscles
which refracted light and which resembled elementary corpuscles of the
virus. The development of such miniscule elementary corpuscles generated
larger transparent light-refracting crystal-shaped corpuscles reaching
2 4 microns in size. These corpuscles were not colored by the method
usually appled for bacteria, but reacted to coloring after being fixed
with alcohol-formalin and to treatment with alkali-eosin. In this case
they were colored red, i.e. they had similar properties with crystals of
yellow jaundice virus of the silkworm and the oak silkworm, but differed
from them in form as w ell as in size.
Thus the experiments showed that artificial inocculation of cater-
pillars with a pure bacterial culture causes formation of specific
crystal-shaped corpuscles which possess properties similar to those of
yellow jaundice crystals. And sowing of crystals in artificial nutrient
media produced invariably manifestation of development of the initial
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kv f li'ail.~. Y 1Q
In connection with this a question confronted us: could not the
observed changes in forms of bacterial cells in the caterpillar organism
and the appearance of crystal-like corpuscles be caused by the change
of form of microbe existence due to changes in the surrounding media?
This hypothesis followed not only from our observations of bacteria
development in the caterpillar organism of cutworm moth-gam-a, but from
observations of other researchers as well who noted repeatedly the double
infection of silkworm caterpillars with cellular as well as virus form of
microbe. Thus according to Nikiforukfs data.,
-92-
(begin p 93)
in Bashkiriia was recorded witk ryellow:'jaundice infection of "unpaired"
silkworm which was developed from silkworm eggs inoculated with Nosema;
in 1944 V. P. Pospelov (6) discovered infection of caterpillars in first
age with Nosema. During their further growth infection with Nosema and
yellow jaundice was observed. V. P. Pospelov points out that flare-up
of the Nosema disease during the second generation of the mulberry silk-
worm in the Ukraine in 1945, occurred also due to mixed infection with
Tosema and polyhedral disease, under which conditions the adult butterflies
contained Nosema and polyhedra but in their eggs were found only spores of
Nosema.
Fig. 3 (p. 92) Change in crystal
in solid nutrient media: A.
substance of crystal obtains
viscous structure of a living
substance; B-crystal-like
corpuscles..
Fig. (p. 93) New formation
of crystals in ba cterial
culture after sowing of
crystal: A--sowed crystal;
B--depression as a result
of thinning of agar with the
crystal; V--newly formed
crystals.
Besides that, Pospelov and Noreiko noticed already in 1929, that it
is possible to inoculate silkworms with yellow jaundice by feeding them
with a culture of yeast Debaryomyces tyrocola con. isolated from cater-
pillars of the "-ionashenka" silkworm which were sick with yellow jaundice.
And at that time it was assumed that the yeastcells in entering the
organism of the silkworm caterpillar change into the virus form of
existance but this transition was not proved by the authors experimentally
under artificial conditions.
-43
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(7) Trans. ay?
Our observations of the development of Bact. Gamma sp. nov. in
artificial nutrient media indicate that in the mentioned bacterial culture
take place also changes in the forms of bacterial cells.
We fixed them directly in the organism of the insulated caterpillar
of the cutworm moth - gamma. Simultaneously appeared in-the bacterial
culture transparent, light-refracting crystal-like corpuscles.
The consistency of these corpuscles which are formed in the cater-
pillar organism as well as in bacterial culture in artificial nutrient
media, is at first viscous. In a fluid drop they have an oscillatory
motion, being in a suspended state, and after a certain time they harden
and take the form of crystals-tetrahedrons which are deposited.
The tetrahedrons which we detected inthe organism of caterpillars
which were inoculated with bacteria and in the bacterial culture itself
in artificial nutrient media did not dissolve in alcohol, ether, acetone
and weak solutions of acids, but readily broke down into elementary
corpuscles in weak solutions of alkali.,
Crystal-like corpuscles-tetrahedrons were formed in the organism
of an insect and under artificial conditions at a 30 - 350 temperature
-93-
(begin p. 94)
as well as at 3 - 5%.
Fiore intensive process of tetrahedron formation in caterpillars
artificially inoculated with bacteria, was observed at 20 - 25?. At
such temperature the breaking-down of a caterpillar organism through
bacteria and formation of tetrahedrons wag observed already 3 - 4
days after the inoculation and at 13 - 17 -on the seventh - ninth day
and later.
At first appeared single specimens of tetrahedrons and with breaking-
down of the organism their number increased. After a complete breaking-
down of the caterpillar organism the amount of tetrahedron is so large
that it cannot be calculated when a regular loop inoculum is taken.
Crystal-like corpuscles-tetrahedrons were formed in all the experi-
ments with pure bacteria culture in organic media rich with albumin, as
well as in inorganic ones with mineral sources of nitrogen. With the
beginning of mass formation of these corpuseles in the caterpillar
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organism as well as in artificial nutrient media, the bacterial culture
frequently lost the intensiveness of growth when sub-cultured as if it
lost its viability.
In a series of experiments the bacterial culture which lost the
capacity of growing in artificial nutrient media was brought into an
active state by passing through a living organism of a caterpillar.
When caterpillars are inoculated artificially with tetrahedrons which were
treated for various tired periods (from 15 min. to 24 hours) with 96?
alcohol, then already 24 hours after the inoculation in the hemo-lymph
of the caterpillars begin to appear normally developed vegetative
bacterial cells.
Soot after sowing of these cells in artificial nutrient media., a
development of the initial bacterial culture became manifest, On the
second day after the inoculation the amount of bacterial cells in the
hemo-lymph of caterpillars increased considerably and a process of
breaking-down of the organism began and on the fourth-fifthcday the
caterpillars were destroyed and a new - formation of crystal-like
corpuscles-tetrahedrons was recorded.
Analysis of the intestines of caterpillars artificially inoculated
with tetrahedrons showed that tetrahedrons pass with food up to the middle
intestine where they undergo changes, break down and at the same time
0 appear bacterial cells.
We Checked under artificial conditions the possibility of obtaining
bacterial cells from crystal-like corpuscles-tetrahedrons, and it was
possible to record definite changes in tetrahedrons. 24 hours after the
sowing some of them began to react to coloring with methylene blue:
they became light-blue colored. After 3 days the number of tetrahedrons
being colored increased considerably; in part of them the center was
colored more intensely. On the fifth day we discovered tetrahedrons in
which the intense coloring was in 8 - 12 spots on the periphery. At
that time no uncolored tetrahedrons were discovered, On t he seventh
day bacterialeells were forming, chains of which looked like a coiled
spiral while tetrahedrons disap-I:eared. Resowing of these bacterial
cells in nutrient media, produced a manifestation of development of
bacteria which were isolated previously from caterpillars of the
cutworm moth-gamma.
On the basis of all the above mentio__ed we came to the conclusion
that the crystal-like corpuscle-tetrahedrons which we discovered inthe
organism of caterpillars, butterflies and eggs of cutworm moth-gamma
seem to present a specific form of bacteria existence.
-94-
0
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(9) Trans. 493
In breeding isolated bacteria under artificial conditions in-,various
nutrient media, together with the crystal-like corpuscle-tetrahedrons
aggregate crystals were formed which, unlike the former did not dissolve
in alkali but instead readily broke down
-94-
(begin 3. 95)
in an albumin media with a weakly acid (p4-6.5) or neutral reaction
into crystal-like corpuscle-tetrahedrons.
In various nutrient media the f orm of crystals was different:
bipyrami.ds, polyhedral rods, prisms, do-decahedrons, polyhedrons and
druses. But in whatever media, we bred the bacteria, the crystal-
aggregates were generated and grew without fail in the bacterial colony
itself or in the film. If the media was unfavorable for the development
of 1 cterial colony, then the aggregate crystals were not formed.
When breeding in solid nutrient media, the crystals which were
generated in bacterial colony grew into the depth of the nutrient media,
frequently forming crystalline particles up to 1 ern. in size.
In liquid nutrient media the, aggregate crystals were generated directly
? in the bacterial film and after it (the film) dropped to the bottom of the
flask they appeared also inside the 'oth in the bacterial residue on
the walls and the bottom of the container,
Studying biological properties of our isolated bacteria in various
artificial nutrient media we established that decrease in temperature down
to 9 - 13?---inhibits and rise to 17 - 22?--speeds up the process of
crystal-formation.
At 30 the development of bacterial culture did not stop but pro-
ceeded extremely slowly. If at 13? and above the clouding of the meat-
peptone broth was observed after 24 hours, formation of bacterial film
after 3 days and formation of drystals 5 days after the inoculation, then
at 3 0 it took place correspondingly on the 8th day, after 12 and 18 days.
In solid nutrient media the process of formation and crystallization
takes longer than in liquid nutrient media. In the first case the
crystallization process of the microbus living substance lasted 55
days, in the second - 22 days.
The temperature factor which influences the intensiveness of
development of the bacterial colony reflects also on the crystallization
-95-
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(10) Trans. 493
0
process of the living substance--the virus. At a 23 0 temperature the
beginning of crystal-formation in the bacterial colony was recorded
on the third day after the sowing of bacteria, at 9o--on the eighth
day and at 5?--on the twentieth day.
In studying the crystallization process in the tncteri_,l film in
liquid nutrient media, we found out that the amount of nutrient
media in the container is of no particular importance but the size of
the surface area of the broth in the container plays an essential role.
The larger the area of the bacterial film, the more crystals are formed.
It was established also that crystal-formation in bacterial culture
is in direct relation to the intensiveness of bacteria development,
to pH and the qualitative composition of nutrient media. At the moment
of intensive crystal-formation in the .cterial film takes place a
change in the pH of the media towards neutralization (in a media with
acid as well as with alkali reaction).
When the pH of the media changed sharply towards acidity or al-
kalinity, bacteria did not develop and crystallization was not observed.
And when the media was changed gradually by the organism itself it
appeared to be capable to change the form of its existence (up) to a
virus and a crystal, With the formation of crystals, bacterial culture
was able to stand a prolonged action of sun rays and high temperature.
Our experiments demonstrated that with the formation of crystals
in the media, pasteurization at 70? and 2-hour sterilization at 120?
(begin p. 96) $
stop the development of the culture, but after a certain time appears
a cellular form of the microbe which generates crystals anew.
-96-
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CD
O
Table 2 (T. 96)
Effect of sterilization of meat-peptone broth inoculated with Loct. Gamma
sp. nov. on subsequent manifestation of development of microbe's cellular
form and new formation of crystals.
Variant of experiment
Manifestation of
development when
froth is resowed
after sterili-
tzatjgn
U) r-.I
?H
U f-~
U
E O U
W 0
rl ~'
CO Cd
G') 0 CC ?ri
rl
Or
0 O
O
CO Cd CH (1)
t i ?k ~t O r-H U
0 () a) cd cd
44 4J,P4_P10
Meat-peptone broth
ino cul^ted with
7/IX
115/IX
i 2/VI
17/
28/
13/
cellular form of
microbe.
Sowing of inoculated
broth in meat-pep-
1948
ro
1948
r.
11949
r._
VII
VIA
VIII
---
tone agar 2 days
after sterilization
(
.
in autoclave at 120?I
_--
---
---
4/VI
4/VI
None
None
Repeat sowing of brothl
in meat-peptone agar
4 days after steri-
lization.
---
6/VI I
u i
n
Sowing of inoculated
broth in meat pep-
tone agar 6 days
after sterilization
8/VI
,I 1
n
Sowing of inoculated
broth in meat pep--
1
tone agar 46 days
20/
25/
after sterilization
9/VI:
VII
VII
Same
p
n
it
i
-96-
of
development
microbe
after sterili-
zation
14pnifestation of
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-96-
In our experiments the artificial inoculation of caterpillars of
cutworm moth-gamma with cellular and virus microbe forms as well as
with crystals of living substance which were isolated from a Incteri al
culture caused their mass infection and:death.
Dissection of diseased and dead caterpillars disclosed a charac-
teristic picture of pathological process which takes place when cater-
pillars die under natural conditions. In cells of hemo-lymph and fat-
albumin body a formation was observed of crystal-like corpuscles-
tetrahedrons and in the cavity fluid of caterpillars was also disclosed
a cellular form of microbe.
We checked the possik t,lity of restoration of microbe life from
crystals isolated from aa.cterial culture in a series of tests in which
we disinfected the crystals superficially (on the surface) with 96?
alcohol and ignited them with the flame of burning alcohol. Experiments
demonstrated that for restoration of life from crystals it is necessary
to create such conditions in the media which would be favorable for a
gradual change of the crystalline substance itself, its transition from
the crystalline to the colloidal state. In the bacterial colony
which developed from a crystal a rather intensive crystal-formation
took place anew. Repeat tests-always produced similar results,
T, ^ e, 3 (P, 97)
mamics in dying off of caterpillars of cutworm moth-gamma in the experi..
ment with artificial inoculation with the microbe of Bact. Gamma. sr). nov.
(1952; experiment conducted on September 11).
Variant of inocu-
lation of cater-
pillars
Control (without in-
oculation).
Th ouch food with
crystals from bac-
terial culture.
Throu`h food with
virus form of
microbe.
j Dead Caterpillars detected
c- 0
>G
G)' MP
I El +-) CO r-- 1 r--I
10 404
r-4 &4
F { H i-i H H' H H' H. fit, (tj
40 10 ' 01 0 0 00 0 0 2
! I I
40 6 3' 14 11L
404 3 1,1!12 9I 1
Through food with cel i. 120" 17 131' 10 13 113
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(13) Trans. 493
ID
eT~
Observing the development process of bacterial colony when aggregate
crystal was sowed into the nutrient media, :?e established the following.
Already 4 hours after sowing of crystal-aggregate in nutrient media,
the agar is liquefied in the spot where the crystal is located. In con-
nection with this a crater-like depression is formed around the crystal
which (depression) is filled T=r!i.th fluid, and the crystal still preserves
its shape-it has no noticeable changes.
On the second day after the sowing, the crystal-aggregate loses its
transparence, becomes dull white and acquires from outside a noncrystal-
line viscous structure. At this moment the viscous mass being formed
consists of minute crystal-like corpuscles-tetrahedrons which possess
properties of liquid crystals very similar to crystals of the yellow
jaundice virus of insects.
With the transition of the crystal into the indicated state its red
coloring with alkali - eosin is possible, which is characteristic for
crystal - like virus corpuscles - polyhedrons which are being found in
insect organisms,
On the third day after the sowin? of the crystal in the place of its
location in the agar starts a formation process of bacterial cells which
are at first roundly, coccus - like in shape and immobile and then stretch,
acquire a rod-like shape and become mobile. The cells which acquired a
rod-like shape reproduce intensely by way of paired division. With the
appearance of the rod-like shape of cells which are mobile, bacteria spread
over the surface of the solid nutrient media. On the fifth - seventh day
after the sozrin of the crystal, aggregate crystals are formed anew.
Bacteria culture from the crystal of aggregate which was generated
in bacterial colony ca-,-, be obtained also in liquid nutrient media. In this
case the generating process of living bacterial cells proceeds consider--
able slower than when the crystals are sowed on the surface of a so'.id
nutrient media.
When aggregate crystal was sowed in solid nutrient media, a bac-
terial colony developed intensively after 48 hours and new crystals were
formed 5 days after the sowing of the crystal, and when similar crystals
were sowed in liquid nutrient media, the intensive development of bac-
terial culture was recorded only after
(begin p. 98)
25 days, formation of bacterial film - after 35 days, new formation of
crystals-after 38 days.
-97-
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(14) Trans. 493
0
This phenomenon which we obseri-ed provid a basis for the assumption,
that in order to obtain the cellular form of the microbe from crystals,
not only a nutrient media is required hit also a direct action of the air,
which a:parentlT- furthers a speedier T,eneration of such a process which
makes a transition possible of the living substance from a crystalline
state to a non-cryrstalline; together with this favorable conditions are
created for formation of the cellular form of the microbe. Correctness
of this is confirmed by the fact that the bacterial colony did not develop
from the crystal directly into the depth of the solid nutrient media.
Table 4, (p. 98)
Effect of recrystallization of the living; substance of the microbe of
B~ct. Gimma sn. nov. on development manifestation of the micr'obe's cell-
ular form (1950)
Variant of Solution of cry;s Date manifestation of development !
Of so- ~. _._ --_.`--
tals and time of soT,in ff ' trin? i 1
C In 30 Sulfuric acid. Sow-
neutralization NA OH 30/VIII s - J - - - - -
In 3O ,;b Sulfuric acid 4.
rieutrilization of solution
NA OTT. SoT-inr~; prior to do-
-posit of crystals. 130/VIII
In 3Ojo sulfuric acid
! !
neutralization T'aOh. Sow- ing of deposited crystals. 30/VIII131/Va l/I.X 2/IX 4?/IX' 8/Ix 8/'i=
In 10j hydrochloric acid
neutralization NaOH.
bowing of Deposited cry sta.I s. 26/VII I ;2q/VII `3%I: i 2E TII L -
In order to test the possibilities of encystment (inclusion) in the
&f tile' ntidr dbe 6f ce-liulAr- form with preservation of its viability`,
we conducted experiments with crystallization of crystals. It was establish-
ed that after dissolving the cryrstals in 10 and 30% solutions of sulfuric
-98-
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%v6
and hydrochloric acid, reactivation of cellular forms of the microbe is
impossible and after neutralization of the gisen solution with a 25%
solution of.alkali, i. e..after? a return depositing of the crystalline
substance., the lat-'-.er is capable to produce cellular form of the mic-: obe
in artificial nutrient media, And the process proceeded in the same
order as when the crystals, which underwent surface desinfection with
alcohol., were sowed.
The oxocriment with recrystallization of crystals which were isolated
from a bacterial culture was conducted with three and six repeats and in
all the cases a similar picture of development manifestation was observed.
The experiment demonstrated that we are dealing here not with a
simple entrapment(?) of microbe cells in a substance being crystallized,
but with a
(begin p. 99)
formati-n in a bacterial colony of a substance which possesses properties
of a living; substance capable for reactivation and development into the
cellular fo--m of microbe.
Thus the living, substance which passed-into a crystalline state is like
a transitory stage. Depending on conditions of the envirorr:nt there can
develor either ay?rocess of formation of albumin substances which have signs
of life, or a process of decomposition as a result of which restoration of
life is possible only under conditions of return depositing of living sub-
stance as crystals.
The results of observations of life restoration process from non-
ce17.ul.ar livin substance of the microbe which changed to the state of the
crystal allow to maintain, that the development. of a non-cellular living
substance of the virus can be carried out only under certain conditions
and when there is a uniformity with them. Outside of that the virus form
can change to crystal form and through that become a lifeless body capable,
however, to retain its structure and to renew the life activity when
necessary conditions are restored.
The phenomenon of crystallization of the living substance gives at
clear example of oneness of the living- and non-living, when between the
two the---e is no i_soassable abyss about which scientists are talking who
occupy ideelistical and metaphysical positions.
The above facts which demonstrate the possibility of transition of
~a-ct.e-:ia into virus and crystal forms in artificial solid and liquid.
-99-
0
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(16) Trans. 493
nutrient media, - confirm fully the cor:rectne,:s of opinions expressed by
D. T. Tvanovskii, N. F. Gamabia, V. A. Krestovnikova, L. T. Fal'kovich and
others on virus as on a result of variabilit`r of forms of existence of the
microbe.
The development process of microbes under artificial conditions w ich
we followed un from bacterial cell to virus form and aggregate crystal and
vice versa from aggregate crystal to bacterial cell, is quite complicated
and requires fur+her more profound stud,;-.
.bli o ranhv
B`
1. Bosh'ian, G. 1% on the nature of viruses and microbes. 14oscow N. 1949.
2. Zhurkov-Verezhnikov, N., MMiaiskii, L., Kalinichenko, L. On non-cellular
3.
forms of life and cell development. Zhurn."Bol'shevik", No. 16, 1950
Ivanovskii, D. :. On two tobacco di sea.ses. Bull. AN. , No. 2. 1892
4. Ivanovskii, D. T, -osaic diseases of tobacco. 1902
5. Lepeshinskaia, 0. B. Development of life processes in the pre-cellular
period. 12d. AN SSSN, 1952
6. Pospelov, V. P. Yellow jaundice of silkworm and control measures against
it. "Doklad; V SKL NIL", V7.-.,-). 9-10, 1946
7. Pospelov, V. P. NN;.icrobiolocicaal method of control of agricultural pests.
"Dokladvr VASIch INTIL", vyP. 7, 1944
8. Sukhov, K.S. on or;-in of viruses. N. 1948
9. Woiks (papers) of the conference of institutes of epidemiology and
microbiology on the problem of changeability of mic-_ obes. Gor'kii, 1949
10. Shchegolev, V. N. Insects w-lich harm field crops. Sel'khozriz, 1937.
All-Union Scientific-Research
Institute for Flax, Torzhok,
Kalinin oblest'.
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(1 Trans. 494
(In full)
Byt
A. Antik
Rautenshtein, Ia. I.
Habliudeniia za lizisom aktinomitseta
pod vliianiem aktinofaga pri pomoshchi
elektronnogo mikroskopa
[Observation of lysis of Actinomycete
under the influence of actinophage with
the aid of an electronic microscope].
Mikrobiologiia 22(1)811-14. Jan/Feb 1953
448.3 M582
(In Russian)
Observations with the help of
an electronic microscope of
1,,. sis of Actinomucete under
the influence of an actinop'hage.
The decomposition process of bacterial cell under the influence of
researchers.
hacteriophaL-e was studied by ;/
inter7-elation bet'-Teen the cell anr'. the phage and of the generatinr,: mechanism
of forms resistant to phages.
But up to the present ti_ e these is no work in the literature with
a description of tl-,he electronic-microscopic picture of the decomposition
of hyeelium of ~~ctinoin,; cotes under the influence of an actinophage, in
spite of the fact that phago-lysis of Actinom cet.s is known since 1934,
when for the first time a work appeared about it by the Soviet scientist
,hitriev (1). Krasit'nikov (21) described the microscopic picture of
dissolving; of colonies of some Actinorcycetes and pro-Aetinomy-cetes as well
as the lysis process of their mycelium as a result of auto-lysis.
In the works by Kriss, Rukina and. Isaev (4) there are data on results
of study with the help of an electronic microscope of the structure of a
normal mycelium of Actinomycetes. In soy-re works on study of actinophages
are Pi1Ten only, photographs of actinonh.a es (7,8). At the same time the
study of the necomposition process of mycelium under the influence of the
phage is undoubtedly of interest for the understanding of some aspects of
The purpose of the present work is the stud- through an electronic
microscope of the deco:.,Tposition process of the. Actinomycete.mycelium under
the influence of actinophage. Act. jlob:isK)orus culture suseotible to
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actinophage and actinophage specific for it, was taken for this cork.
In chosing a media for breeding Actinomycete and for phago-lysis
we decided on glucose.-asparagine media of the following composition:
glucose--- 1.Og., asparagine- 0.059. , K2 I~t'01 - 0.5g, Id' aCl - 0.59-, corn
extract - 0.1 - 0.2g., distilled water - 100 ml. Sterilization of media
was carried out by filtering thi ou.g h a Seitz's filter. The indicated
media is favorable for the growth of Actinomycetes and for manifestation of
lytic action of actinophage. The advantage of this media consists in the
fact that in it the growth of Actinomycote and the process of its lysis
under the influence of actinophage is slower than in media rich in albumin,
which makes the observations of sincle stages of this process easier. The
breeding was carried out under conditions of depth growth in flasks placed
in a _ocl..r (agitator) at a moo tempcratur:. Under these conditions, as
observations showed, phago-lysis proceeded more co "letely than with sur-
face brooding.
Experiments with phago-lysis w^r e conducted in two variants. In some
tests actii.Q -phase was introduced into t' e media. simultaneously with. spores
of Actinomycete; in others .. actino-phage was added to the already grown
24-hour culture of Actinomycete. As a control served the culture of
.Actinomycete beinj; grown in the same media but without actino-phago. Dia-
lysis w o, car- icd out throurrh collodion films by the method. described by
Kriss, Berezova and ZolkoverW (3) and lasted usually 15-16 hours.
Fixation of preparations for electronic - microscopic observations
was done by dossication. I For this pug nose the material from flasks bein
studied was transfer- ed with a, loop directly on small collodion films
which were stretched on special small mztallic -nets used for preparing of
electronic-m_ic--oscop-? c pre-oarations.
(Begin p. 12) Preparations prepared. by the indicated method were
dried in the air at room temperature and then placed for dialysis.
Results of exnerimonts
i orphological peculiarities of actino-phage used in our experiments
for the lysis of Art. ql.obisnorus culture are shown in fig. 1.
C na tion for fiauros 1-10. 1) Actino-phage of the Act. mlob_i_s 2orus
culture. Dusting with Nichro o. 2) Snores of the Act. .,lobisuorus
culutre. 3) Germination of Act. r:i.obis;.poru,s spores. 4) Same. 5)
Normal 24-hour cultu:. e of Act. alobisnorus. 6) Act. r?i_obi~.snorus
spore germinated in presence of actino-phage which underwent a l:;sis
after the formation of a short myceliuim, 7) iiyceliw;. grown from a
8) NI,y-
spore iii a media with actino-pha ;e at the age of 2 4 houl?s S.
celium of Act. r?lo,i sno:r?rs after one hour cxoos zJ e to actino-phaf e.
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In one section of the hypha the lysis has commenced. 9) Mycelium of
Act, rlobi.sporus after one hour exposure to actino-phago. 10) Same.
It is clearly seen from the presented photograph that a particle of
this actino-phage consists of an oval-shaped head and a tail. The head of
the actino-phage is not homogeneous but consists of a lighter in color)
central part and of two more dense corpuscles at the poles.
The size of actino-phage's head when dusted with Nichrome is about
30-35 w*i in diameter along its long axis the length of the tail is about
twice as long as that of the head.
Looking, through an electronic microscope at the spores of the A_Ot. .
rrlohismoru.s it is seen that they are predominatly cylindrical in shape (fig.
2), before r?ermination the spo ?es swell and become rounded (fig. 3) or
retain their cylindrical shape (fig. 4).
In fig. 5 a normal mycelium of the culture is shown at the age of 24
hours. It is seen that at this are it has a homogeneous structure with single
lishter sections.
In introducing into the r,.lucose-asparagine media the spore material
of Actinonrrcete simultaneously with the actino-phage the following picture
is observed; at first the spores swell and germinate as in the control, i.e.
in the media without actino-phage.
Part of the spores undergoes a lysis immediately after germination and
formation of a short mycelium (fig. 6).
Another part of the spores continues its growth for some time after
the nermina.tion and forms an entire ;r normal branching, myrcelium. In fig. 7
is shoulm such a culture at a 21 hour age.
Mycelium grown in media with an actino-phage can reach various sizes
and be exposed at different stages of its development to the ly is effect
of actino-phage.
The lysis of mycelium which grew from spores in the presence in the
media of actino-ph.-,.L e, proceeds in the same way as the lysis of a nature
my*celium to which the phage was added 24 hours after the sowing of spores.
Therefore the description of mycelium lysis for the two cases is given to-
gether.
Changes in the grown mycelium of Actinonr;rcete to which actino-phage is
added during breeding in glucose -asparagino media and which are noticeable
through the electronic microscope, can be usually detected an hour after
introduction of actino-phage.
-3-
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0
0
C
These changes begin in single sections of each hypha not covering at
once the hypha as a whole. It can be noticed how in the s;-)ot of the hypha
where the lysis starts being manifest, at first comes a certain thickening
of it, the protoplasm becomes more dense and separates slightly from the
wall. This is seen in fi . 8 where the state of.thc mycolium section is
shown an hour after the exposure to actinophage.
In fir-. 9 and 10 are presented picturds of the state of mycelium which
can be observed after one hour exposure to: the phage and which indicate fur-
ther development of the lysis process.
In single sections of the hyphae the:-protoplasm loses its homogeneity,
spots appear with a noticeable granularity---(fig. 9). By the same time there
are single hyphae in which the lysis process has developed still further.
In fig. 10 is shown the state of such a mycelium. Clearly seen is a sharp-
ly exuressed nuclear granularity and presence of sections with a transparent
content.
The character of further development of the lysis process can be judg-
ed (begin p. 13) from fig. 11, which demonstrates the state of some hyphae
after a 2 hour exposure to actino-phage. It is clearly seen how the section
of mycelium of a normal aspect changes sharply into a.bysod section, in
which the wall is already destroyed and the content broken down into a
shapeless mass.
Cantions for fi-::res 11-7B . 11) Hyoha of the Act. globispor:?us my-
celium after a 2 hour exposure to actino-phage. 12) The Act.. rrlobi-
snoi us mycelium after a 3 hour exposure to actino--pha e. 13) ilycel-
ium at the age of 27 hours in a media without actino-phage (control).
14) iyceliurn of Act. lobisnorus after a 4 hour exposure to a.ctino-
phage. 15) Sane. 16) Single hyphae of the Act..rlobis-aorus mycelium
after a 4 hour exposure to actino-phage. 1'7) Same after a 5 hour
exposure to actino-phage. 18), Same after an B hour exposure to actino-
phage.
After a 3-3 hour exposure to actino-phage many hyphae are in a state
of complete decomposition; in some of them are -retained single small myceli-
un sections with a granular content which is in varying stages of decorm-
position (fig. 12).
The mycelium in the control flasks (without actino-phage) by this
time retained fully its homogeneous structure (fig. 13).
After a 4 hour exposure to actino-phar.'e the amount of fully lysed
hyphae increases, but among thorn are found single hyphae which have fully
retaif.;4 the normal mycelium (fig. V?).
Figures 15 and 16 show the state of single hhiphae of the Actinomycete
culture after a. L A-2 hour exposure to actino-phage.
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In fig. 15 we see parts of two dif 'event hyphae; in one of them the
lysis prooess terminated at one and with a. tear in the wall and a break
down into r shapeless mass and the other part still retained single
sections of mycelium of normal. aspect.
In fig. 16 are illustrated three hyphae which are in various stages of
lysis. On one of them (b) remains a smallsection with normal mycelium
-which changes into a lysed section; another' lrpha (a) is almost completely
lysed, and the third hypha (d) contains extremely charactoristic granular
inclusions on the nature of which it is difficuit to pass a judgement.
After 5 hours of contact between the actino-phage and the rhyccli-um, in pre-
parations can be observed, together with 13rsed hyphae and hyphae completely
broken do-v*n into a shapeless mass, also si~:gle sections of hyphae which
retained :.itirely their, normal aspect. Fig. 17 demonstrates the state of
some hyphae after a 5 'aour exposure to actino-phage. It is seen clearly in
this fia_?ure how a normal section of one hypha terminates ahrumntlsr and is
sharply separated from the following entirely lysed part of the same h;pha.
And after an 3-1 hour exposure of mycelium to actino-phage, when the
number of the completely decomposed h ro1ae is already quite considerable,
there are single complotely preserved sections of hyphae (fig:. 18).
M
Longer observations of the lysis process of Actinomyceto indicate
that together with a complete decomposition of some hyphae an appearance of
a young mycelium at the expense of e secondary groirth can be observed. Ile
secondar yr gro*..