Professional Documents
Culture Documents
4293
by Z. P I ~ I K R Y L and V. VAN(~URA
Dept. Soil Microbiology, Inst. Microbiology, Czechoslovak
Academy of Sciences, Prague
KEY WORDS
SUMMARY
The release of substances from wheat roots was found to be directly related to the growth of the root
system. Plants whose root system did not grow released almost no exudates.
When exudate concentration in the vicinity of the roots was lowered by frequent replacements of
the nutrient solution or by a simultaneous cultivation of exudate-utilizing bacteria, the release of
exudates was enhanced. In axenic wheat cultures, the amount ofexudates during a 12-day cultivation
with 2- or 4-day intervals between medium replacements represented 50% of root dry weight and
12% of whole plant dry weight.
Wheat plants cultivated in the presence of the bacterium Pseudomonasputida released up to double
the amount of exudates compared with axenic variants.
INTRODUCTION
69
Plant cultivation
Wheat seeds of the cultivar lliichovka were surface-sterilized for 20 min by 0,1% HgCI 2 sup-
plemented with 0.1% detergent Tween 80 (Lachema). The seeds were then pregerminated for 24 h
under sterile conditions on wet filter paper and tested for another 24 h for sterility in Petri dishes with
meat-peptone agar. The plants were cultivated under axenic conditions in glass cylinders placed in
500 ml flasks (cfFig. 1) with 50 ml nutrient medium. A total of 6 plants were cultivated in one flask.
The nutrient solution contained 0.333 g Ca(NO3)2, 0.040 g MgSO4, 0.063 g KNO3, 0.060 g KH2PO,,
0.00025 g Fe2(504)3, 0.0001 g ZnSO4,0.0001 g H3BO3 in 1000 ml distilled water. After sterilization,
the pH of the solution was adjusted to 7.2-7.4; during the subsequent cultivation, fresh aliquots of the
medium were used to replace the cultivation liquid under aseptic conditions at 2, 4 and 6-day
Fig. 1. Experimental setup for the cultivation of plants under axenic conditions. 1 - wide-neck
Erlenmeyer flask (500 ml), 2 - glass tube (length 400mm, diameter 35 mm), 3 - gauze, 4 - cottonwool
stopper, 5 - nutrient medium.
ROOT EXUDATES OF PLANTS 71
intervals. The cultivation was carried out in a phytotron at 20 ~ C; light (intensity 7500 lux) was
applied at 16-h intervals interspersed by 8-h dark intervals. A total of 30 plants were measured and
weighed in each experimental variant. The differences were evaluated statistically using the t-test at a
significance level p = 0.01. In variants in which the growth of plants was studied in the presence of
bacteria growing on the roots and in their vicinity, the nutrient medium was inoculated with the
suspension of bacteria of the genus Pseudomonas putida, strain K 2.
Exudate determination
The biomass in the nutrient medium was determined as dry weight on membrane filters (pore
diameter 0.6p.m) after a previous removal of plant cells and other contaminations by filtration on
filters with pore diameter of 4 p.m. The dry weight of bacteria on the roots was calculated by
multiplying the number of bacteria determined after root crushing in a blender by the average weight
of one bacterium determined by a simultaneous assay of bacterial count and bacterial dry weight in
the nutrient solution. The n u m b e r of bacteria was determined by plating on Petri dishes with agar
medium. 14
RESULTS
The basic data on the growth of wheat plants in aqueous media under axenic
conditions and with different medium replacement intervals during the first
twelve days of cultivation are summarized in Table 1. Neither the length nor the
weight of above-ground parts of the plants were statistically significantly affected
by the different frequency of renewal of the medium. The growth of both
parameters was linear almost till the end of the period under study (Fig. 2). On
the other hand, the growth of the root system slowed down after about six days,
depending on the frequency of medium replacements. The length of the root
system in the variant with 2-day replacement interval was, after a total of 12 days
of cultivation, significantly (about 20%) lower than in other variants, the weight
being also lower by 10-12%. This fact may be apparently ascribed to more
favourable conditions in the variant with higher frequency of medium replace-
ments.
The total amount of exudates (Table 1) was directly proportional to the
frequency of medium replacements and thus to the rate ofexudate removal from
the medium. With 2-day and 4-day intervals of medium replacements the
72 Z. PI~IKRYL AND V. V A N ~ U R A
~0
o=
r~
o
r~
"5
,.J
r
r~
b-
8
O
ROOT EXUDATES OF PLANTS 73
4ot A I-i 3O
I ShootsI ,~1
301 /
-/ /
2C
Shoots //o
//
,//
~ii [ / 6 I /o
1C
] o// ......_....4-68
/Q 0._...--0
/ r/ Roots E" - / Roots
T 0
/ J 8/
. . . . . . . . .
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Days after planting
Fig. 2. Growth o f wheat roots and shoots A length in cm, B - weight in m g under axenic
conditions in aqueous media replaced at 2-, 4- and 6-day intervals (denoted by numerals on
individual curves).
amount of exudates was higher by 40%, with 6-day intervals by 16% as com-
pared with the culture in which the medium was not replaced for 12 days.
The time course of exudation (Fig. 3) is very similar to that of the root system
growth. Figs. 4 and 5 show thus the total amount ofexudates in dependence on
the length or weight of the root system in plants in which the nutrient medium
was replaced at 2-day and 4-day intervals. The linearity of these relationships
indicated that, at least in the initial phase of wheat growth, the exudation was
directly associated with the growth o f the root system and was directly propor-
tional to it.
Hence, the linear parts of the dependences may be described by regression
equations that give the following numerical values and regression coefficients
Fig. 4:
2-day replacements y = 0.153 x - 0.296 r = 0.998
4-day replacements y = 0.121 x - 0.135 r = 0.997
Fig. 5A:
2-day replacements y = 0.019 x - 0.112 r = 0.999
4-day replacements y = 0.016 x - 0.078 r = 0.995
74 Z. PI~IKRYL AND V. VAN~URA
" 1,5
m
t,._
(3.
C
0
t,..._
1,0
O1
E
e-
(1)
"o
)<
w 0,5
/
O
I i i I i i
2 4 6 8 10 12
Days after planting
Fig. 3. Amount of wheat root exudates (mg C per plant) under axenic conditions. Nutrient medium
was replaced at 2-, 4- and 6-day intervals (indicated by numerals; N-variant with no replacement).
ROOT EXUDATESOF PLANTS 75
./
A
c(01 5 m
I
Q.
Q.
C
0
.,Q
r
14i
0 1,0
E
r
, i
x
u.i 0,5
0
/
/
J
J
i I I I I I I I I I I I I I I
5 10 15
Length (cm)
Fig. 4. Relationship between exudation amount (rag C per plant) and the growth of the wheat root
system (cm) at 2- and 4-day replacements of nutrient medium.
Fig. 5B:
2-day r e p l a c e m e n t s y = 0.226 x - 0.112 r = 0.999
4-day r e p l a c e m e n t s y = 0.196 x - 0.086 r = 0.994
A 2/./
1,5
c
m
O.
C
0
o 1,C
0
O~
E
/
.c= oe
"0
x: 0,5
/
/ /
/ /
. . . . s'o . . . . 1 o' 5 10
Fresh weight (mg) Dry weight (rng)
Fig. 5. Relationship between exudation amount (mg C per plant) and fresh (A) or dry (B) weight
(mg) of wheat root system at 2- and 4-day replacements of nutrient medium.
nutrient medium. The exudate carbon represents about 2% wet weight and
about 20% dry weight of roots in axenically grown plants. If referred to the
weight of the whole plant, the exuded carbon corresponds to 0.5% wet and 5%
dry weight.
If the exudates were utilized for the growth of bacteria inoculated into the
nutrient medium and were thus continuously withdrawn from the medium, the
exudation was higher by 60-80% as compared with axenic variants (cf com-
parison of values in Table 1 and Table 2). The growth of plant shoots was not
appreciably affected by the presence of the bacterium Pseudomonasputida K 2 in
the nutrient medium and on plant roots. The root system was smaller by about
10%. The total amount of biomass of the bacteria, for which the root exudates
are the only source of carbon, was markedly lower in the variant with no
replacements of the nutrient medium as compared with other variants. This is in
keeping with the lower exudation found in the same variant of an axenic culture.
ROOT EXUDATES OF PLANTS 77
e.
9" ~ t'q t~ r
8 b
t
r t'q t~
r r t'q
r
tt~ t.~ oo
t'q r ~
O
"~ ..-
r~
t~
~e
r~
r t'q r
t'-q r r
z
78 Z. PI~IKRYL AND V. VAN(~URA
0,8 2 014
2
R
~' 0,6 0,3 ~~
'10
0,4 0,2 ~
c
E
1 N
f,
0,2 0,1 ~
0 2 4 6 8 10
Days after planting
Fig. 6. Kinetics of wheat root exudation (mg C per plant per day) and bacterial mass (mg dry
weight per plant per day) at 2-day replacements of nutrient medium. 1 - plants cultivated in a sterile
solution, 2 - plants cultivated in the presence of growing cells of Pseudomonasputida (calculated from
the bacterial biomass), 3 - bacterial mass in nutrient medium.
ROOT EXUDATES OF PLANTS 79
The kinetics of exudation in the variant with 2-day medium replacements are
shown in Fig. 6. At the initial stage there was almost no difference between the
exudation under axenic conditions and in the presence of microorganisms in the
nutrient medium and on roots. Only on the 4th day, when a maximum of
exudation was attained under axenic conditions, the effect of nutrient removal
from the medium by the bacteria was demonstrated by a marked increase in
exudation which persisted even at the stage of the general decline of exudation at
the end of the experimental period.
Fig. 7 shows the relationship between the total amount of organic carbon
liberated by the roots (exudation values in Table 3) and total amount of carbon
incorporated in root biomass in the same period. The relationships may be
described by the following regression equations:
3,0 2e
-ff
o
u
2,0
E
C
o ~'~ 1
U) o~
"1o
x
w 1,0
I I I I I I I I I I I I I I I I I I I
The difference in coefficients denoting the slopes of the two curves indicates that
the amount of organic carbon exuded from the roots of axenic wheat plants was
about half that incorporated in the root biomass. The variant with the Pseudo-
monasputida culture showed about the same amount of carbon in the roots and
in the exudates.
DISCUSSION
cultivated under field conditions from the early tillering stage to flowering.
Johnen and Sauerbeck 5 found the same amount of carbon exuded from roots
and utilized by soil microorganisms only during the first 30 days corresponding
also to the tillering stage. At later stages the amount of carbon accessible to soil
microorganisms exceeded the harvested root mass 2.5-fold.
The mechanism of exudation of substances by plant roots is not yet fully
elucidated. It seems probable that all known mechanisms of transport of sub-
stances through cell membranes participate in the translocation of the substances
into the root-free space. From there, the substances diffuse into the environment.
This is indicated by the data on the increase of exudation from roots into the
environment when the permeability of the cell membranes is temporarily dis-
turbed by a moisture or temperature stress 6, ~2.15,16 and by our present data on
the effect of a change in the concentration gradient ofexudates between the roots
and the environment. When the ambient concentration of substances released by
the roots of wheat plants is lowered either by frequent renewals of the nutrient
medium or by a simultaneous cultivation of bacteria with the plants, with
exudates serving as the only carbon source, the exudation increased substan-
tially. The effect of a continuous removal of the exuded substances by the
bacteria that had been found earlier 17 was found even when the external exudate
level was lowered by frequent medium replacements. This could be due to
methodological differences in carbon determination in axenic cultures and in the
presence of microorganisms. The direct analytical determination of carbon in
the medium includes only partially the poorly diffusible mucilage substances
adhering to the root surface. However, these substances may be utilized for
growth of the microorganisms living directly on the roots and, in the indirect
assay of exudates from the growth of the bacterial biomass, they may be
determined along with other substances that diffuse readily into the nutrient
medium.
According to Rovira ~2 the exudates of older parts of the roots contain
60-70% of substances diffusing readily into the medium.
On the other hand, the tip portion of the root about 5 cm in length, which
exhibits the highest exudation, releases only 30% of diffusible substances, the
rest being of mucilage character. The most substantial distortion of data should
thus occur when the exudation is determined during the first 4 days of growth
when the longest primary root attains about 7 cm in length. Yet the comparison
of axenic cultures with those in which the exudates were utilized by bacteria
showed no appreciable differences in exudation during this period. On the other
hand, the change in diffusion conditions should be the most conspicuous in the
exudation from the oldest parts of the root system. Indeed, the exudation
82 Z. PI~IKRYL AND V. VAN~URA
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