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Growth of noninfected and PIasmodiopkova bvassicae infected cabbage callus in
culture1
P. H. WILLIAMS,M. N. REDDY,AND J. 0. STRAND BERG^
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Departrtret~tof Plant Pathology, Utrivet~sityof Wisconsin, Mndison, Wisconsitr
Received February 17, 1969
, 1969. Growth of noninfected and PlastrroWILLIAMS,P. H., REDDY,M. N., and S ~ A N D B E RJ.G0.
diophorn brnssicae infected cabbage callus in culture. Can. J. Bot. 47: 1217-1221.
Noninfected cabbage callus and Plast~~odiophorabrassiccre infected callus isolated fro111 clubroot
galls were maintained for over 40 successive transfers o n Murashige-Skoog's medium modified to
contain 0.5 mg/l of a-naphthalene acetic acid. Infected callus if transferred at 7- to 12-day intervals
grew rapidly, doubling its dry weight about every 3 days, whereas noninfected callus doubled its dry
weight about every 5 days. Approximately one-third of the cells in infected callus contained the parasite in various stages of its life cycle, ranging from small vegetative plasmodia to mature resting sporangia. Sporangia isolated from callus were viable and produced clubroot inoculated on cabbage seedlings. When a high percentage of the plasmodia in infected cells in any portion of a callus underwent
sporogenesis, the callus growth slowed and the tissues became brown. By transferring only actively
proliferating callus a high percentage of plasmodia could be maintained in the vegetative condition.
Infected callus resembles closely gall tissue from natural clubroot both cytologically and chemically
and thus should be a useful material for studying parasitism and the processes of hypertrophy and
hyperplasia in a contaminant-free system.
Introduction
A lack of success in culturing obligate fungal
parasites on artificial media has prompted the
development of tissue culture techniques for the
study of these organisms. Callus tissue infected
with a single parasite provides a means for examining the physiology of parasitism without
the influence of contaminating organisms. A
comparison of the nutritional requirements of
infected and noninfected callus tissues could
make it possible to ascertain the nutritional
factors required to grow the obligate parasite
in vitro.
Although there have been a number of reports of obligate fungi being grown in monoxenic culture with their host tissues (1, 1S), few
of these have led to productive programs on the
nature of obligate parasitism or to the culture of
the obligate parasite itself. AS in the work of
Nozzolillo and Craigie (18) with Puccinia helianthi Schw. on Helianthus annuus L. or in the
work of Heim and Gries (7) with Erysiphe
cichoracearum D.C. on H . annuus, the fungus
killed the callus and subsequently died out, before many successive transfers of the culture
lThis work was supported in part by the National
Kraut Packers Association, in part by the American
Cancer Society, Grant No. IN-35G-13, and in part by
Public Health Service Grant A1 4149. Published with the
approval of the Director, Wisconsin Agricultural Experiment Station Project No. 2007.
2Present address: Central Florida Experiment Station,
Sanford 32771.
could be made. Milholland (14) was unable to
maintain Puccinia granzinis var. tritici infected
wheat callus in culture beyond a single transfer.
The most successful cultivation of an obligate
parasite on host callus has been that of Cutter
(3) for Gymnosporangium juniperi-virginianae
Schw. on Juniperus virginiarza L. Infected calluses were maintained up to 8 years by successive transfers and eventually led to the isolation
of the fungus on artificial medium. Turel and
Ledingham (22) succeeded in producing abundant sterile mycelium and spores of Melampsora
lini (Pers.) Lev. on the cotyledons of flax in
organ culture and Nakamura (17) has maintained
contaminant-free Peronospora parasitica on turnip root explants in culture. With the exception
of the systemically infected juniper callus grown
by Cutter, most of the fungi in tissue culture
grew superficially on the callus and eventually
the noninfected tissues outgrew the invaded
cells.
Unlike many of the filamentous obligate fungal parasites, Plasmodiophora brassicae Wor.
remains entirelv within the host cells where it
stimulates a callus-like gall on the roots and
hypocotyls of various crucifer species (2). Previous cytochemical observations indicated that
the Dresence of the ~arasitein crucifer cells was
an important factor in the hyperplastic and
hypertrophic response leading to gall formation
(23). Because of the superficial similarity between clubroot gall tissue and tissue culture
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1218
CANADIAN JOURNAL OF BOTANY.
callus, it appeared that a study of P. brassicae
infected callus in monoxenic culture would enable cytochemical and physiological studies on
the fundamental processes underlying gall formation and parasite growth. Comparisons of
various parameters for growth and metabolism
of infected and noninfected cabbage callus with
those already reported in the literature for
natural clubroot galls would determine the
validity of using tissue culture callus for study
of the clubroot disease. In addition to providing
a material for physiological work, infected callus
would be a contaminant-free source of P. brassicae plasmodia (11) suitable for attempted in
vitro culture of the parasite. This study is a more
detailed account of the previously reported work
on the monoxenic culture of P. brassicae in
cabbage callus (21). Recently Ingram (9) also
has reported on the growth of P. brassicae infected crucifer callus in tissue culture.
Materials and Methods
Infected cabbage callus was isolated from young
actively growing clubroot galls and maintained on a
synthetic medium. Clubroot galls were produced on
cabbage plants, Brassica oleracea var. capitata L.,
Jersey Queen, grown in P. brassicae infested soil using
the methods described previously (25). Infected roots
were removed from the soil 25-30 days after the plants
were transplanted into infested soil and then washed
thoroughly in running tap water. Young white clubs between 2 and 3 mm in diameter were selected and further
washed by shaking them for several minutes in sterile
water in a stoppered flask. Clubs were transferred to a
57, chlorox solution in a petri dish for 5 rnin, cut into
4- to 5-rnm sections, and then transferred to 207, chlorox.
After 5 rnin in 20% chlorox the sections were rinsed for
10 min in 10 changes of sterile water and placed individually into test tubes containing 5 ml of the tissue culture
medium.
The chemically defined medium of Murashige and
Skoog (MS) (16) as modified by Linsmeier and Skoog
(12) was further modified by using a-naphthalene acetic
acid (NAA) (0.5 mg/l) instead of indole-3-acetic acid
(IAA) and by adding kinetin (6-furfural aminopurine) at
1.0 mg/l (21). The medium was adjusted to pH 5.8 with
NaOH before it was autoclaved and its pH was 5.6 after
autoclaving.
The tissue explants were placed in the dark at 22-24
OC. Tissues contaminated with bacteria or fungi were
discarded. After 7-10 days, tissue pieces showing cell
proliferation were transferred to fresh medium in test
tubes and as callus developed it was excised, examined
for the presence of the parasite, numbered, and transferred to flasks of the culture medium. Actively growing
callus was divided and explants transferred to fresh MS
medium every 7-12 days. Only cream or light-colored
callus was transferred. Cultures were maintained in the
dark at 22-24'C in 125-1111 Erlenmeyer flasks or 6-oz
VOL. 47, 1969
prescription bottles containing 25 ml of the MS isolation
medium. T o each bottle, four or five explants were
transferred each weighing 3&45 mg (dry weight).
Noninfected cabbage callus was obtained from hypocotyls of noninfected plants in the same way as described
above for infected galls, or from hypocotyls of germinating Jersey Queen seed as follows. Cabbage seed was
surface-sterilized for 10 min in 107, chlorox, then washed
thoroughly in five changes of sterile water. Seeds were
then transferred to flasks or bottles (four per bottle)
containing 25 ml of Hildebrandt's D tissue culture
medium (8). After 10-15 days on this medium the small
seedlings began to develop callus from their roots,
hypocotyls, and cotyledons. After 25-30 days callus
originating from the hypocotyl and root regions of the
seedling was excised, numbered, and transferred to fresh
medium. Rapidly growing clones were transferred every
7-12 days on D medium. After 2 4 weeks the rapidly
growing clones were transferred to MS medium supplemented with 500 mg/l of casamino acids (Difco) and
2.0 mg/l of 2,4-dichlorophenoxyacetic acid (2,4-D) in
place of the NAA. This enriched medium was better for
the initial transfer of noninfected callus from D medium
because callus which was transferred directly to MS
medium became brown and died. To avoid this transfer
shock callus was brought to the MS medium over a
period of two or three transfers by eliminating the casamino acid in the first transfer and replacing the 2,4-D
with NAA in the second or third transfers. After the
noninfected tissues had become adapted to MS medium
they were grown and maintained in the same way as
infected callus.
Growth rates of infected and noninfected callus on
MS medium were established by determining the fresh
and dry weights of 10 tissue pieces from two bottles at
specified intervals. For comparison, callus derived from
pith of Nicotiarza tabacst~tzL. var. Wisconsin No. 38 was
grown and sampled in the same way as cabbage callus.
These experiments were repeated at least 4 times.
lnfected and noninfected tissue culture callus was
examined cytologically at different times between 2 and
12 days after explants were transferred to fresh medium.
Pieces were fixed in formalin - acetic acid - alcohol
(FAA) and dehydrated in a tertiary-butanol series. After
standard paraffin embedding and sectioning techniques
sections were stained in safranin and fast green (10). Ten
sections from each of three different callus pieces taken
at different times were observed for parasite development and host cell response. The number and volume of
cell nuclei and nucleoli from infected cells and from noninfected cells in normal callus were estimated from diameter measurements, assuming that the nuclei and
nucleoli were spherical. The volume of infected and
noninfected cells was estimated from length and width
measurements, assuming the cells were cylindrical.
The number of cells per milligram of infected or noninfected callus was estimated by hydrolyzing 20-30 mg
of tissue in 2 ml of 107, chromic acid for 8-12 h and
then counting the separated cells with a hemocytometer.
Infected and noninfected callus, 8 days after transfer
to fresh medium, were analyzed for a number of metabolites by the identical procedure outlined previously for
the separation of metabolites in clubroot galls (24).
Callus tissue was washed in distilled water to remove
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WILLIAMS ET
AL.:GROWTH
excess medium and agar, then frozen in liquid nitrogen.
Frozen pieces were powdered, lyophilized, dried at 80 OC
for 24 h, and then stored at room temperature over CaC12.
Samples of dried tissue (10 mg to 20 mg) were analyzed
for total N by a Kjeldahl procedure (24), for total lipids
by the quantitative sulfuric acid charring method (20),
and for total sugars with the anthrone reagent (4). The
quantity of starch was determined by the iodine method
of Hassid (6), protein with the Lowry reagent (13), and
free amino acids by the method of Yemm and Cocking
(27). Four classes of phosphorous compounds, inorganic,
acid labile, lipid phosphate, and nucleotide phosphates,
were separated and extracted by the procedure of Smillie
and Krotkov (19) and analyzed for phosphate by the
Fiske and Subbarow method (5).
T o determine the viability and infectivity of P. brassicae, resting sporangia produced in tissue culture 25- to
30-day-old callus pieces which had turned brown were
minced in a petri dish under aseptic conditions with a
sterile razor blade. The released sporangia were suspended (105 spores/ml) in a dilute salt solution (20) in
5-ml vials. Three-day-old Jersey Queen seedlings grown
on water agar from surface-sterilized seed were placed in
the vials containing sporangia. After 1-week root hairs
were examined for infection and seedlings were transferred t o sterilized quartz sand and watered with a balanced nutrient solution. Thirty days later the seedlings
were washed free from sand and observed for clubroot.
1219
OF CABBAGE CALLUS
doubled its weight every 5 days. Tobacco pith
callus grew at a similar rate as noninfected
cabbage callus.
Infected callus had a loose, irregular shape,
and white to cream-colored growth (Fig. 2B)
whereas noninfected callus was yellow and had
a firm consistency (Fig. 2A). Infected callus
averaged (4.5 rt 0.8)103 cellslmg fresh weight
and contained about 8.670 dry matter. Noninfected callus averaged (16.2 5)103 cellslmg
fresh weight and contained 12.1y0 dry matter
(Table 11). Frequently, 7-12 days after transfer,
small sectors of an infected callus piece began
to darken and slowed in growth whereas other
sections remained light-colored and continued
to proliferate rapidly. Tissues which were not
transferred to fresh media at regular intervals
usually completely darkened and became necrotic in 3-4 weeks. Occasionally a tissue piece
darkened within a few days of transfer without
any apparent reason. Frequently necrosis of a
whole infected tissue piece was preceded by the
production of many small rootlets and profuse
root hairs.
Results
Between 1 and 5% of the P. brassicae infected
cabbage root explants produced continually
growing callus on tissue culture. Most of the
explants died as bacteria within the tissues
multiplied. Individual clones of tissue isolated
from the same root explant had different growth
rates. The gross morphological characters of
certain clones changed with repeated passage on
the culture medium. Some clones continued as
undifferentiated callus, whereas others produced
many thickened structures resembling poorly
differentiated roots. By selecting only undifferentiated tissue for transfers, the differentiating
property of some clones could be eliminated.
Root-hair-like structures were also observed on
some infected and occasionally on noninfected
callus. These root-hair-like structures could also
be eliminated by selective transfer of callus
tissue. By transferring fast-growing pieces of
callus to fresh media at 7- to 12-day intervals,
several rapidly growing clones have been maintained for more than 40 transfers. Figure 1 depicts the growth rate of infected callus clone,
JQ-2, compared with a rapidly growing noninfected clone. Infected callus doubled its dry
weight every 3 days whereas noninfected tissue
--
P. B R A S S I C A E
I N F E C T E D CABBAGE
T I S S U E CULTURE
/I
>
TISSUE CULTURE
0
2
4
GROWTH
6
8
10
12
PERIOD IN DAYS
FIG. 1. Increase in dry weights of noninfected and
Plasmodiophora brassicae infected cabbage callus grown
o n a chemically defined medium.
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1220
CANADIAN JOURNAL OF
The various stages of parasite growth and host
cell response found in natural clubroot galls
(23) were also observed in tissue culture. Infected callus had numerous sites of meristematic
activity with many cells containing small plasmodia. Characteristic "Krankheitsherde" were
common and when the parasite occupied many
cells in a localized area, host cell division slowed
while plasmodia enlarged and eventually sporulated. An average of 34% of the callus cells
contained the parasite in various stages of
development. Darkening and necrosis of the
tissue were usually accompanied by sporulation
of the parasite. As was found in clubroot galls,
host cell responses such as nuclear and nucleolar
enlargement (23) and increased starch content
(24) were restricted to the parasitized cells.
Nuclear and nucleolar volumes increased an
average of 4.5 and 23.8 times, respectively,
compared to cells from noninfected callus (Table
I). As was found previously with clubroot galls
(24) total nitrogen, free amino acids, protein,
total lipids, and starch were all higher in infected
than in noninfected callus tissues (Table 11). Of
the various phosphate compounds, lipid and
nucleotide phosphates were slightly higher in
TABLE I
A comparison of size of cell, nucleus, and nucleolus in
noninfected and Plosmodiophora brassicae-infected cabbage callus tissue in culturea
Factor
measured
Noninfected
Cell size, p 3
7260
Nucleus size, p 3
195.5
Nucleolus size, p 3
1 .6
a
Infected
Increase in
infected
61879
878
38.8
8.5
4.5
23.8
Data are the averagc of 30 randomly sampled cclls.
BOTANY.
VOL. 47, 1969
infected tissues than in noninfected callus. Unlike in clubroot gall tissue, sugar levels were
lower in the infected callus than in noninfected
callus (Table 11).
Resting sporangia collected from monoxenic
cultures of P. brnssicne were viable as numerous
plants became infected and produced clubroot
galls 30 days after having their roots dipped in
spore suspensions.
Discussion
The comparative ease with which P. brassicae
infected cabbage callus was isolated and maintained in tissue culture on a chemically defined
medium suggests its potential usefulness for the
contaminant-free study of obligate host-parasite relations. Cytologically and ultrastructurally the parasite and the parasitized tissue
culture cells are similar to those in natural
clubroot galls (24, 26). Metabolites shown to
increase in infected tissue culture callus also
resemble those known to be high in natural
clubroot galls (24). Although the changing
amounts of metabolites in growing callus were
not followed, it is unlikely that callus would
show the sharp increases in starch, sugars, and
nucleic acids that are known to occur when
cabbage seedlings become infected with P.
brassicae, mainly because the callus is a heterogenous mass of cells at different stages of infection. Some cells contain mature resting
sporangia whereas others contain young vegetative plasmodia. In the case of young cabbages
(24) the rapid buildup of starch and sugars in
the gall reflected a response of the host to invasion by the parasite. Unlike in the cabbage plant
TABLE I1
A comparison of various metabolites in noninfected and Plasmodiophora brassicae infected cabbage callus tissue cultures at 8 days after transfer to fresh
medium
Metabolite
Dry wt. (mg/g fresh wt.)
Total nitrogen
Protein
Free amino acids
Total sugars
Starch
Total lipids
Total phosphate compounds
(a) Inorganic P
(b) Acid-labile P
(c) Lipid phosphates
(d) Nucleotide P
Noninfected,
mg/g dry wt.
Infected,
mg/g dry wt.
Increase in
infected, mg
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FIG. 2. T h e appearance of noninfected cabbage callus 16 days after transfer (A) and (B) Plaa~~oriiopl~ora
brasricne infected cabbage callus 8 days after transfer.
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WILLIAMS ET
1
I
I
AL.: GROWTH
about one-third of all the cells in tissue culture
callus are continuously infected.
The localization of large amounts of starch
in the infected cells in callus is similar to that in
the cabbage root h ~ p o c o t ~galls.
l
As it does in
the clubroot galls, the starch in infected callus
cells disappeared upon sporogenesis
of the
parasite (24). Unlike natural galls the infected
callus had a lower sugar content than noninfected callus. Noilinfected callus had 2 times as
much sugar per gram dry weight as noninfected
h ~ ~ o c o t (24)
~ l s Or as infected
The
tively low levels of sugars in the infected callus
may reflect a rapid metabolizing of sugars since
the respiration of infected callus tissue doubles
in the presence of excess exogenous glucose or
sucrose (unpublished data). Conversely the high
Sugar contellt of noninfected tissue c0~1ldresult
from an accumulation of sugars in tissues which
are unresponsive metabolically to exogenous~y
supplied sugars (unpublished data).
Infected callus can be maintained in an active
log phase Of growth through many transfers by
frequent transfer and selection of actively growing sectors of tissue. The rapid growth of infected
to nOninfected
suggests that the parasite is providing some form
of growth
in addition to that
in the medium. Although it is unknown whether
the parasite is directly promoting growth by
providing growth factors, or whether it is stirnulating- the host to do so, the tissue culture system
seems to be feasible for investigating the problem.
Acknowledgments
The authors acknowledge the assistance of Dr.
N. T. Keen with some chemical analyses and of
Eugene Herrling and Steve Vicen with the figures.
1. BRIAN,P. W. 1967. Obligate parasitism in fungi.
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caused by Plasmodiophora brassicae Woron. Commonw. Mycol. Inst. Phytopathol. Pap. 3.
3. CUTTER,V. M. 1959. Studies on the isolation and
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4. FAIRBURN,
N. J. 1953. A modified anthrone reagent.
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5. FISKE,C. H. and S u n n ~ ~ o w
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66: 375-400.
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33-36. I n R. J. Smith, J. N. BeMiller, and M. L.
1221
OF CABBAGE CALLUS
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~
y
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RANDALL,
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R. D. 1962. Callus tissue from wheat
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and SKOOG,F. 1962. A revised medium
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J. 0. 1968. Lipid metabolism in
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J. O., WILLIAMS,
P. H., and YUKAWA,
Y. 1967. Monoxenic culture of Plasmodiouhora brassicae with cabbage tissue. ~ h y t o ~ a t h o l o g y(Abstr.)
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and MCNABOLA,
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P. H. and WALKER,J. C. 1963. Races of
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27. YEMM,E. W. and COCKING,E. C. 1955. The determination of amino acids with ninhydrin. Analyst,
80: 209-21 3.
,
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