Effect of Bacterial Wilt on Uptake and Translocation of Phosphorus, Sulphur, Calcium, and Manganese in Alfalfa Plants

Zbl. Bakt. Abt. II, Bd. 130, S. 367-386 (1975)

[Research Institutes for Plant Production, Prague -

Ruzyne, Institute of Plant Protection]

Effect of Bacterial Wilt on Uptake and Translocation of Phosphorus, Sulphur, Calcium, and Manganese in Alfalfa Plants Lubomir Taimr, Anna Kudelova, Vaclav Kudela, and Eva Bergmannova With 3 figures

Summary Under the influence of bacterial wilt 32 P, 35S, and 54Mn uptake pel' alfalfa plant decreased by 28.69 to 46.03 % in both investigated cultivars, the reduction of uptake being significantly higher in the susceptible cultivar Kasticka than in the resistant cultivar Hodoninka. Following infection, 45Ca uptake significantly decreased in the resistant cultiva,r only, while in the susceptible one it was even slightly increasing. 32p, 35S, and 54Mn transport to the overground organs waS restricted after infection which led to accumulation of these elements in the roots. In the susceptible cultivar Kasticka, 45Ca concentration in the overground parts decreased only slightly as a result of infection. Therefore, 45Ca concentration was about equal in overground organs of infected plants of both the resistant and the susceptible cultivar. According to the autograms the first symptoms of the disease, detecta,ble on the overground organs, corresponded with the absence of investigated radioisotopes in these tissues. At more ad· vanced stages of the disease the presence of radioiwtopes was limited to the ['cot and stalk or to the root only.

Zusammenfassung Luzernepflanzen (Medicago sativa L.) del' Sorten Hodonfnka und Kasticka wmden im Keirn· blattstadium mit Corynebacterium insidiosum (McCulloch) Jensen, dem Erreger del' Bakterienwelke, inokuliert. Nach 30tagiger Inkubation wurde markiertes KH232P0 4 , Na 235 S0 4 , 45CaCI 2 und 54MnCI 2 zur Nahrlasung, in der sich die infizierten und als Kontrolle dienenden Pflanzen befanden, zugegeben. 15-29 Tage nach Zugabe del' markierten Verbindungen wurde die Radioaktivitat pro Pflanze sowie die spezifische Aktivitat in den Wurzeln und den oberirdischen Organen bei infizierten Pflanzen und bei den Kontrollen radiometrisch bestimmt. Aul3erdem wurde del' Transport del' markierten Verbindungen in den Pflanzen auch autoradiographisch verfolgt. Gesunde Luzernepflanzen speicherten 32p, 35S und 54Mn vor allem in den \Vurzeln, wahrend 45Ca in den oberirdischen Organen gefunden wurde. Gesundc Pflanzen del' resistenten Sorte Hodoninka wiesen im Vergleich zur empfindlichen Sorte Kasticka einen statistisch gesichert haheren Gehalt an 32p und besonders an 45Ca pro Pfla,nze a,uf. Die Aufnahme von 35S wa,r bei beiden Sorten ungefahr gleich hoch. Unter dem Einflul3 der Bakterienwelke war die Aufnahme von 32p, 35S und 54Mn pro Pflanze bei beiden Sorten durchschnittlich urn 28,69-46,03 % niedriger, wobei diese Verminderung bei del' empfindlichen Sorte Ka,sticka statistisch gesichert gral3er war. 45Ca wurde nach der Infektion nm durch die resistente Sorte Hodoninka in vennindertem Ma,J3e aufgenornmen, wiihrend die 45Ca_ Aufnahme bei der empfindlichen Sorte Kasticka soga,r etwas anstieg.

368

L. TAIMR u. a.

Unter dem EinfluB der Infektion wurde der Transport von 32p, 35S und 54Mn in die oberirdischen Organe behindert, was eine starkere Speicherung dieser Elemente in den Wurzeln zur Folge hatte. Bei der empfindlichen Sorte Kasticka sank die 45Ca·Konzentration in den oberirdischen Organen nur geringfiigig, so daB sie bei beiden Sorten ungefiihr gleich groB war. Aus den Autoradiogrammen geht hervor, daB das Erscheinen der ersten Symptome der Erkrankung an den oberirdischen Organen mit der Abwesenheit von Radioisotopen in diesen Geweben zusammenfallt. Bei in fortgeschrittenen Stadien der Erkrankung befindlichen Pflanzen waren die Isotope auf die Wurzel und den Stengel oder nur auf die Wurzel beschrankt. Folgende Hypothesen wurden aufgestellt: 1. Flir die Entstehung des Syndroms der Bakterien· welke bei der Luzerne ist eine Dysfunktion der Leitblindel von groBerer Bedeutung als die Storung der Saugkraft in den Wurzelzellen. 2. Der Kalziumtransport aus den Wurzeln in die oberirdischen Organe vollzieht sich auBerhalb des Xylems, wahrscheinlich liber den Apoplast. 3. Dem Kalzium kommt in den Abwehrmechanismen der Luzerne gegen die Bakterienwelke eine wichtige Bedeutung zu.

Bacterial wilt of alfalfa belongs to the group of diseases called vascular wilt. The common symptom of these diseases is a disintegration of the vascular system in many angiosperms. The pathogenes, different taxonomically remote microorganisms, bacteria, and fungi attack the xylem part of the vascular bundles. Their presence in the vascular system of the host manifests itself by profound changes in metabolism. These changes find their symptomatic expression in blocking and discolouration of the vessels, wilting and dessication of the overground organs and, eventually, the precocious decay of the plant. From the complex of metabolic changes in plants, invaded by bacterial wilt, major attention has been paid until now to the changes in the content of sugars, nitrogen compounds, activity of hydrolytic and other enzymes, phenolic compunds, growth regulators, and toxins. Only a limited number of investigations has so farbeen devoted to the water relations and to nutrient uptake of infected plants (SADASIVAN 1965). The scope of the present study was to investigate the following questions, using the radioisotope tracer method: 1. To what extent bacterial wilt affects the uptake and translocation of some biogenous elements; 2. do possible changes in the nutrient uptake and distribution, brought about by the disease, manifest themselves to the same extent in all investigated elements or only in some of them; 3. do there exist differences between a susceptile and a resistant cultivar in uptake and topographic distribution of different elements. In the present report, uptake of 32P, 35S, 45Ca, and 54Mn was investigated.

Material and Methods 1. Plant material

Two aJfalfa (Medicago sativa L.) cultivars were used in the experiments: Kasticka, which is very susceptible to bacterial wilt, and Hodoninka, exhibiting a certain degree of resistance. The plants were grown in glass vessels filled with quartz sand moistened with HELLRIEGEL'S nutrient solution. Prior to sowing the vessels with the sand were autoclaved at 2.5 atm. for 20 minutes. Three days old alfalfa seedlings were planted in the vessels, thus prepared on August 23,1972. The vessels with the plants were tra,nsferred to a greenhouse where temperatures fluctuated between 22 and 26°C during the day and between 15 and 19°C during the night. 2. Inoculation of the plants The plants were inoculated on August 28, 1972 at an age of eight days. Three isolates of Corynebacterium insidiosum (McCulloch) Jensen were used for inoculation. The density of the inoculum corresponded to a concentration of about 3.7 X 106 bacterial cells in 1 ml. of sterile distilled water. The method used for inoculation on the cotyledons was described by KREITLOW (1963).

Effect of Bacterial Wilt on Uptake and Translocation of Phosphorus

369

For studying the 32p, 358, and 45Ca u.ptako, 50 plants of each cultivar, placed in one vessel , were inoculated. 54Mn uptake waS inves tigated in 20 incculated plants of tho cul t ivar K9,stick a. The same number of plants served a.s control (Table 1). 3. Application of the labell ed s ub stan c e s 30 days after inoculation wator solutions of Na 235 S0 4 , KH232pO.\, 45Ca.CI~, and 54MnC1 2 , r esp., were a,dded to the nutrient m edium . 0.5 m!. of the respective solution was applied near to the root base of each pla.nt by means of a. pipette. Tho specific activity, th e volum e of the applied solutions, 9,nd the rad ioactivity per vessel and per plant ar e given in Ta.ble 1 for th e ~ ingle labelled compounds. 4. D e t er mination of uptak e a nd t.rans location of th e r a dioi so top eH in the plant s The plants were harvested a nd analyzed 15 to 29 days after application of the la belled compounds. The processing and the dates of analyse~ of t he single va.riants a re quoted in Table 1. The plants wer e taken out of the sand, and t h e root,; wer e carefully rinsed in a st,ream of water. The ha rvested material was divided into two repli cations, consisting of an identical number of pla.nts (Table I). The overground parts were separated from the r'oots and the fr esh and dry weight (after dryi ng at 60 - 70 °C for 24 h.) was determined in both replications. 4.1. Determination of 32.Phosphot'Us Fresh roots and overground pat·ts were twice homogenized with trichloroacetic acid. After cent rifugation the joint supel'1latants we re brought to a volum e of 10 m!. 4 m!. aliquots of the extra.ct were shaken 5 minutes with a mixture of 2.5 m!. benzene, 2.5 m!. isobutanol, 0.4 m!. 5 M sulphmic acid , 0.5 m!. 10 % ammonium molybdate, and 0.1 mi. wate!'. Thereafter, the samples were a llowed to stand until the two phases containing the inorganic a nd organic phosphorus, resp. , were comple tely separated. 0.5 ml. samples were withdrawn from both ph ases a nd t he ir radioactivity m easured on planchets in a Tesla m et h anflow window 2 n counter (window thickness 0.20 mg. /cm 2 ). The dead time of the appara.tus waS 5.4 {lsec. Radioactivity of both th e organic and inorganic phase WaS ta,ken into account in evaluting 321' uptake and translocation . 4.2. D atermino,tion of 35·sulphur Fres h roots and overground parts wer e extracted for 2 minutes with boiling methano!' After cooling they were homogenized and ex tracted again. The volume of the com bined extracts was brought to 2 ml. by evaporation in vacuo at a temperature of 40 °C. Radioactivity was measured in 0.5 m\. of th e concentrate in the counter doscribed above. 4.3. D etermina.tion of 45·ca.lcium Dry ovet'ground parts a.nd r oots wer e mineralized in a a.ppropriate volume of concentrated nitric a.cid, corresponding to the weigh t of t h e sample (10 m!. per g. of dry weight) and in half the volume of perchloric acid. The samples were mineralized on a sand bath, and their volume was reduced to 1- 2 m!. After cooling the volum e was brought to 10 m!. by di stilled water. 0.5 m!. sa.mples were measured in 10 m!. of the liquid scitillatol' SLE 31 (Spolana. Neratovice) in a Mark·I scintillation ounter (Nuclear Chicago Liquid Scintillati.on Computer) at a t emperature of 2 aC. 4.4. D et ermination of 54·manganese Dry overground parts and roots wore mineralized on a sand bath fir st in concentrated nitric a cid (25 m!. /g . dry weight). After the samples had turned yellow, 5 times less perchloric acid was added.

Fmther mineralization took place at a modera te temperature until wh ite fume s ceased developing. Then a small a.mount ofnitl'ic a,c id was a dded , and the samples were heated until complete decolouri· zation h a d taken place and brough t t o a volume of a.bout 1 m!. Mter add ing 5 m!. of distilled wa.ter th e samples were boiled a.nd brought to pH 7 with 4 N sodium hydroxide. The sheer liquid was transferred to a 10 m!. mea.sming vial ami distilled water was add ed. 0.5 m!. samples were counted in 10 ml. of liquid scintillator SLE 31 in a Mal'k-I counter. 4.5. Autoradiography Along with the analyses of t,he pla.nt material autoradiogra.phy of rando mly selectEd plants at different sta.ges of the disease, as well as of the control plants was p erformed. The plants were fixed by h eat (by ironing). Complet ely dry plants were put into casettes with Ilford X·ray film. Plants 25

Zbl. Bakt. II. Abt., Bd. 130

370

L.

TAIMR

u. a.

Ta.ble 1. Schema of the experiment, investigating the effect of infection by Corynebacterium insi. La.belled compound

Cultivar

Date of planting

Kasticka

Experimental variant

Date of inoculation Number of (age of plants) vessels and of plants per vessel

Infected plants

Aug. 28, 1972 (8 days)

1 a 50 plants

Control plants Hodoninka eN

"'0>" M

...w

Infected plants

Aug. 28, 1972 (8 days)

~ bI) ~

0

>1

Control plants

...>1


Infected plants

...<1l<1l

Control plants

~

Hodonimka

"d <1l

'a ;:

Kasticka

...w>1
'a "d

Hodoninka

'9w

:>, cO

"d

M

Kasticka

Infected plants

Infected plants

1 a 50 plants 1 a 50 plants

Control plants

eN

Kasticka

1 a 50 plants

Aug. 28, 1972 (8 days)

1 a 50 plants 1 a 50 plants

Aug. 28, 1972 (8 days)

1 a 50 plants 1 a 50 plants

Aug. 28, 1972 (8 days)

Control plants

1 a 50 plants

1 a 50 plants

Infected plants Control plants

Aug. 28, 1972 (8 days)

1 it 50 plants 1 a 50 plants

Infected plants Control plants

Aug. 28, 1972 (8 days)

1 a 20 plants 1 it 20 plants

containing 35S and 45Ca were in direct contact with the surface of the film and were exposed 4 weeks. Plants containing 32p were covered with a membrane and exposed 6 days. 5. Measuring methods and eval ua tion of the resul ts

In the methan·flow counter the samples were counted 2 X 1 minute, in the liquid scintillation counter 2 minutes. The background count waS taken 2 x 5 minutes. The operation of the methan· flow counter was controlled and corrected by measuring RaD and 90SI' reference sources, as well as standards, prepared in the laboratory from the solutions of known specific activity, which were applied to the experimental plants. The results of the measurements in the methan-flow counter were corrected for the decay of the isotope. The results obtained with the Mark-I counter did not require any correction for dead time as the resolving time is practically zero in this apparatus up to 7 millions counts/minute.

Effect of Bacterial Wilt on Uptake and Translocation of Phosphorus

371

diosum on uptake of labelled nutrients on two alfalfa cultivars Collection of the plants:

Application of labelled compound: Date of application

Specific Volum0 activity of (mI.) appI. solution (ftCi/mI.)

ftCi per vessel

ftCi per plant

Sept. 27, 1972 (plant age 38 days) 12.33

25

308.25

6.125

Sept. 27, 1972 (plant age 38 days) 10.00

25

250.00

5.000

Sept. 27, 1972 (plant age 38 days) 9.15

Sept. 27, 1972 (plant age 38 days)

0.50

25

10

228.75

5.00

4.575

0.250

Date of collection (age of plants)

Oct. 12, 1972 (15 days following application; plant age 53 days)

Oct. 12, 1972 (22 days following appli. cation; plant age 60 days)

Oct. 26, 1972 (29 days following application; plant age 67 days)

Oct. 26,1972 (29 days following app!. : pI. age 67d)

1st repli- 2nd replication: cation: Number of collected plants

Number of collected plants

23

23

24

24

20

20

22

22

23

23

21

21

23

23

25

25

25

25

23

24

26

26

24

24

14 16

The results of the measurements, expressed in counts/min./I. plant, were obtained by converting the radioactivity of 0,5 mI. samples to the total extracted volume which corresponded to the activity of the total number of experimental plants in one replication. After dividing by the number of plants per replication, the values for radioactivity in counts Imin./I. plant were obtained. The specific activities (concentration) of the radioisotopes in the overground organs and roots express activity of the total extracted volume in counts/minute, converted to 1 g. of dry weight. The results were statistically evaluated, using the analysis of variance for double-way classification. The cultivars represented one factor, the infected or healthy plant material the other one. As two replications (except for the experiments with 54Mn) were available for each combination of factors, it was also possible to test the significance of interactions. Besides, the significance of differences between the aVill'age values in the single classes was evaluated, using the STUDENT's t-test.

25*

KH232P0 4

Kasticka

45CaCl2

Both cul t ivars (x )

Ho don inka

Kasticka

Bot h cul t ivars (x)

Hodoninka

Cultivar

Labelle d com pound

16,8 14 .75

Whole p lant

18,934 .0 10,977 . 25 5,83 7.5

p lant

Roo ts Ove rg round par t

~'bo le

10,752 .5 8 , 181.5

1l, 46 1. 0

6,809.25 4 ,651.75

13,331. 5

6,956.0 6 , 375 .5

9,590.5

14,695.5

W h ole p lant

Ro ots Over ground part

6,66 2.5 2 ,928.0

11 ,202 .0 3, 4 93.5

2,335.0

2,260.25

W h ole p lan t

Ro ots Overground part

578 .75 1, 756 . 25

522. 0 2,098.25

2,502.0

:3 , 192.0

W h ole p lant

Ro ots Overg round par t

72 1. 5 1, 780.5

66 1.5 2,530. 5

2 , 168 .0

2, 048.5

W h ole p lant

Roots Overground p ar t

4 36 .0 1,732 .0

x

Infecte d p la n ts:

3 82 .5 1,666. 0

Control plants x ( = 100)

Ro ots Ov erground p art

Part of the p la nt

68. 16

62. 03 79.69

70.4 1

64.69 77 .93

65.26

59.48 83 .81

89. 11

110.87 83 .70

78. :38

109 .07 70.36

105.83

11 3 .99 103 .96

%

±

- 31. 84

- 37.97 - 20 .31

- 29.59

- 35. 31 - 22.07

- 34.74

- 40. 52 - 16.19

- 10.89

+ 10. 87 - 16.30

- 2 1. 62

+ 9. 07 - 29.64

++ 11 ++

+ + -1 + + +

++ ++

+ + +++ +

+ + ++

+ + +++ +

+

+++ +

o f c ontr o l

+ 13.99 + 3.96 + 5 .83

%

Ta ble 2. Radioactiv ity of infecte d and control p la n t s of alfalfa in counts/min. /pla n t (x ). Differences exp r essed as per cents of infected plants as compared t o controls

t..J

?'

P

~

~

r

-:! t-o

~

Explanat ions:

MMnCl 2

Na2 35S0 4

783.0

4,1l4.00

W h ole plant

Whole p lant

2,156.75 1,95 7.25

Roots Overgrou nd part

439.0 344. 0

2, 901.0

4 ,068.0

W h ole p lant

Roo t s Overgr ound pad,

1, 656.0 1, 245.0

1, 783 .5 2,284.5

Roots Overground p art

530.0

309 .0 221.0

2,573 .00

1,5 15.50 1,05 7. 50

2 ,24 5.0

4,160. 0

W hole p lant

1 ,375.0 870.0

2,530.0 1,630. 0

Roots Ovel'gro und p a r t

"' = signifance at a = 0 .25 0.10 0.05 0.025 0 .0 1 0.005

++ + ++ ++++ ++ ++ + ++++ + +

K asticka

B oth cul t ivars (x)

H odoninka

K ast icka

- 37.46 -- 2H.61 - 35.76 - 32.31

70.3\) 64.24 67 .6\)

- 29.73 - 4 5 .97

- 28.6\)

7.15 -45.50

- 4 6 .03

-45.65 - 4 6.63

62 .54

70.27 54.03

71. 31

92.85 54.50

53. 97

54.35 53.37

++++ ++++ + + "' + + T

+ + ++

+ !+

+++ + +

++ + + + ++

,....,

t>j

~

-:t

~

...,o ::: en

::r"

w '"d

o

::r"

'"d

g,

:;

ct"

'o·"

0" Q

'n

::I

f-3 ~

-

S ;'""g

:;

o

~

:::::: .....;!

~

"::l .

"..

Q

'"

t;j

o,....,

~

:=t Q

KH232P0 4

Kasticka

45CaC1 2

Both cultiva.rs (x)

Hodoninka

Kasticka

Both cultivars (x)

Hodoninka

Cult ivaI'

Labelled compound

O/R

Roots (R) Overground part (0)

O /R

Roots (R) Overground part (0)

O/R

Roots (R) Overground part (0)

O/R

Roots (R) Overground part (0)

O/R

Roots (R) Overground part (0)

O/R

Roots (R) Overground part (0)

Pa.rt of the plant

0.38145

1429,928.0 450,953.0

0.5299

946 , 326.5 484,033.5

0.2320

1913,529.5 417 , 872.5

2.6191

77,283.75 177,772.75

2. 2332

99,250.0 195,729.0

3.0050

55,317.5 159,816.5

Control plants x ( = 100)

0.240575

1669,629.25 397,466.0

0.31255

1636,647.0 510,478.5

0.1686

1702,611.5 284,453.5

1.451525

100,567.75 142,324.25

1.4232

106,850.0 145,266.0

1.47985

94,285.5 139,382.5

x

Infected plants:

63.07

116.76 88.14

58.98

172.95 105.46

72.36

88.98 68.07

55.42

130.13 80.06

63.73

107.66 74. 22

48.41

170.44 87.21

%

of control

- 36.93 +

+ 16.76 - 11.86

- 41.02 +

+72.95 + + + 5.46

- 27.64

- 11.02 - 3 1.93 +

-44.58 +

+ 30. 13 -19.84+ +

- 3 6.27

+ 7.66 - 25.78 + +

- 51.59 +

+ 70.44 + -12.79

%±

Table 3. Specific a ctivity of roots and ove rground parts of infected and control plants of alfalfa. in counts/min./g. of dry weight (x). Differences expl'essed a s per cents of infected plants a s compared to controls

C;.:>

?'

...~ ~ F

~

~

-l

Explanations:

a4MnCI2

Na 235 S0 4

OIR

Roots (R) Overground part (0)

OIR

Roots (R) Overground part (0)

OIR

Roots (R) Overground part (0)

OIR

Roots (R) Overground part (0)

+ = significance a.t ()(. = 0.25 0.10 ++ 0.05 +++

Kasticka

Both cultivars (x)

Hodonfnka

Kasticka

0.4330

66,857.0 28,947.0

0.5460

279,659.75 148,422.75

0 .6208

303,245.5 178,068.5

0.4712

256,074.0 Il8,777.0

0.1973

78,546.0 15,500.0

0.27365

359,420.5 88,162.0

0.2305

455,257.5 98,699.5

0.3168

263,583.5 77, 624.5

45.57

117.48 53.55

50. 12

128.52 59.40

37.13

150.13 55.43

67.23

102.93 65.35

- 54.43

+ 17.48 - 46.45

- 49.88 +++

+ 28.52 -40.60 ++

- 62.87 + + +

+ 50.13 + - 44.57 + +

- 32.77

+ 2. 93 - 34.65

en

-.::r

~

~

5

~

~

o -,

~ o· ::l

reo

::l

~

>l-

~

~

~

~

[. e. ~ g

b:I ~

g,

~

i;l ~

t;:j

376

L.

TAIMR

u.

a.

It waS decided to indicate the degree of significaece of values, even of these are not significant at the current level (IX = 0.05), the levels IX = 0.10 and a = 0.25 being also quoted. Although values surpassing the 10 or 25 percentage point are not significant, in single Cases they provide evidence for the real existence of an ascertained phenomenon when they 3,re repeatedly found in analogical determinations, as waS the case in the present report.

Results 1. Uptake of n u trien t s in infected and control plants Infection of alfalfa plants by the pathogen of bacterial wilt, Corynebacterium insidiosum, markedly affected the uptake of labelled elements from the nutrient solution. Analyses carried out 15-29 days after application of the isotope proved the plants of both investigated cultivars, inoculated at the stage of cotyledons to which the labelled compounds were applied, followed by a 30-days' incubation period, to contain 28.69 to 46.03 % less 32P, 35S, and 54Mn, resp., as compared to healthy plants. When the average values obtained from both cultivars are taken into account, this reduction is significant (at iX = 0.005) in 32p (by 31.84%) and in 35S (by 37.46%). In the susceptible cultivar Kasticka the reduction in 32p and 35S uptake, brought about by the infection, was more pronounced than in the resistant cultivar Hodoninka. While in the latter the 32p uptake was reduced by 29.59% (iX = 0.025) and the 35S uptake by 28.69% (iX = 0.05), the respective values for the cultivar Kasticka are 34.74% (iX = 0.025) for 32p and 46.03% (iX = 0.01) for 35S (Table 2). 45Ca uptake was affected in a different fashion. In the susceptible cultivar Kasticka the expected reduction in Ca uptake was not found, on the contrary, infected plants exhibited even a 45Ca content higher by 5.83 % than the healthy ones. However, this increase is not significant. On the other hand, infected plants of the resistant cultivar Hodoninka contained 21.62% less 45Ca than non-infected ones. This reduction is significant (iX = 0.25). More detailed data are quoted in Table 2. 2. Translocation of nutrients in the plant Distribution of the investigated elements in the plants and changes in translocation, brought about by infection, were estimated by comparing the values of radioactivity, counted in roots and overground organs and converted to I g. of dry weight (specific activity) in healthy and infected plants (Table 3), as well as according to the radioautograms, prepared from healthy and infected plants. 32P, 35S, and 54Mn accumulated at a considerably higher degree in the roots than in the overground parts in both healthy and infected plants. The calculated values of the ratios of specific activity between overground organs and roots (OjR) show how many times the specific activity of the overground organs is smaller as compared to that of the roots (Table 3). The results obtained from multiplying these values by 100 may be considered to represent the percentage of specific activity in the overground parts in comparison with the roots (= 100). According to the data thus obtained, the reduction of specific activity of 32P, 35S, and 54Mn, resp., in the overground organs of non-infected plants, as compared with the roots, ranged from 23.30 to 62.08%. The value for 32p amounted to 23.30% in the cultivar Kasticka and to 52.99% in the cultivar Hodoninka, for 35S the respective values were 47.12 % in Kasticka and 62.08 % in Hodoninka, for 54Mn 43.30% in Kasticka. In infected plants, the difference between roots and overground parts still increased, obviously because of a restriction in nutrient transport to the overground organs. If the OjR values of noninfected plants are considered to be 100, those of infected ones are reduced by 27.64 to 62.87 % (Table 3). Thus, owing to the infection, the 32P, 35S, and 54Mn transport

Effect of Bacterial Wilt on Uptake and Translocation of Phosphorus

377

from the roots to the overground organs decreased by about one third to more than a half. When taking into account the average values obtained from both the cultivars, the reduction of the 32p (-36.93%) and 35 8 (-49.88 %) transport is significant at the level of iX = 0.25 and 0.05, resp. In the cultivar Kasticka, the decrease in the 32p (-27.64%) and 35 8 (-32.77%) transport was not significant. In Hodoninka, the reduction of the 32p transport (-4l.02%) was significant at the level of iX = 0.25 and that of 35 8 (-62.87%) at the level of iX = 0.05 (Table B).

Fig. 1. Alfa.lfa. pla.nts (at left) a.nd their positiva autoradiogra.ms (a.t right) a.fter application of 32p to the nutrient medium. On the top: control plants. On the bottom: pla.nts infected by Corynebacterium insidio8um.

378

L.

TAHIR

u. a.

I Fig. 2. Alfalfa plants (at left) and their positive autoradiograms (at right) after application of 35 8 to the nutrient medium. On the top: control plants. On the bottom: plants infected by Corynebac. terium insidiosum.

Translocation of 45Ca in the plants of both cultivars was quite different from that of the other investigated elements, as it resulted from a comparison of the O/R values. In noninfected plants the specific acitivity (concentration) of 45 Ca was 2.23 to 3.00 times higher in the overground organs than in the roots (Table 3). The specific activity of 45Ca was reduced in the overground organs of both cultivars as a result of infection, and an increase in Ca accumulation took place in the roots. Reduction of the OjR values in infected plants was significant at the level of ex = 0.25 in the cultivar Kasticka (-51.59%) and in the average values from both investigated cultivars (-44.58%), but not in Hodoninka (-36.27 %). The autoradiograms revealed the topographic localization of the investigated elements (Figs. 1, 2, 3). The first symptoms of the disease in the overground organs, yellowing and withering of the leaves from the margin, correspond with the absence of radioisotopes in the tissues. In more advanced stages of the investigated disease, radi oactivity is confined to the root and stalk or to the root only.

Effect of Bacterial Wilt on Uptake and Translocation of Phosphorus

379

Fig. 3. Alfalfa pla.nts (at left) and their positive autoradiogra.ms (at r ight) a.fter application of 4·Ca to the nutrient medium. On the top: control plants. On the bottom: pla.nts infected by Oorynebacterium insidio8um.

3. Effect of cultivar in nutri e nt uptake and translocation Besides the above-mentioned differences between both investigated cultivars, it was observed that in non-infected plants of the resistant cultivar Hodoninka the 32p activity was higher by 28.84% per plant on the average, as compared with the susceptible cultivar Kasticka . This increase is significant on the level of 1X = 0.05. The 35S uptake was about the same in non-infected plants of both cultivars, the lower uptake (by 2.21 %) in Hodoninka being not significant. However, following infection the 35S uptake was clearly higher in the resistant cultivar Hodoninka (by 29.22 % per plant on the average at the level of 1X = 0.25), as compared with the susceptible

KH232P0 4

Wh ole plant

45CaCl 2

Ove rground parts

Roots

\Vhol e plant

Ove rgl"ound parts

Roots

Part of th e plant

Labelled compound

18,934.0 13,331.5 16,132.75 10,752.5 6,956.0 8,854.25 8,181.5 6,375.5 7,278.5

14,695.5 9,590.5 12,143.0 11, 202.0 6,662.5 8,932.25 3,493.5 2,928.0 3,210.75

C + I(x)

Control plants (C) Infected plants (I)

C + I(x)

Control plants (C) Infected plants (I)

C + I (x)

2,155.5

1,699.0

C + I (x)

Control plants (C ) Infect e d p lants (I)

2,530.5 1,780.5

1,666.0 1 ,732. 0

691.5

409.25

C + I (x)

Control plants (C) Infected plants (l)

661.5 721.5

382.5 436.0

2,847.0

2,108.25

C + I (x)

Contwl plants (C) Infected plants (I)

3,192.0 2,502.0

x

\-

168.97

226.69

234.1 9 217.74

99. 13

95.99 104.41

132.86

128.84 139.01

126.87

+ + -+- -+

-+ + T -+ + +

+,-

0.87

4.01 + 4.41

+ 126.69 + + +

+

32.86 +++ +

28.84 39.01

26.87 + +

5 1.89 2.80

68 .97 + +

72.9 4 65.48

35.04 ++ +

55.82 +++ 15.41

+ 134.1 9 +++ + 117.74 ++

+

+

+

+

+

+ +

+ +

172.94 165.48

151.89 102.80

+

+ +

% ± of Kast.icl
135.04

155.82 115.41

%

Cul tivaI" H odoninka

2,048.5 2,168.0

Cult ivaI" Kasticka x ( = 100)

Control plants (C) Infected plants (I)

State of the plant

Table 4. Radioactivity of control a.nd infected plants of alfalfa in counts/min./plant (x). Differences expressed as p er cents of the culti var Hodoninka as compa red to the cult iva r Kasticka

~

?'

>=

~

~

:;;:

-'3

r'

0

00

<:..:i

Explanations :

Na. 235S04

+ ++ +

+ ++

-\-. =

++

+I (x)

+I (x)

C

+I (x)

Con trol plants (e) Infected pla.nts (I)

C

Control pla.nts (C) Infecte d plants (I)

C

Control pla.nts (C) Infecte d pla.nts (I)

s ignificance at ex = 0.25 0.10 0.05 0.025

Over ground pa.rts

Roots

Whole plant

1,250.0

1,630.0 870.0

1,952.5

2,530.0 1 , 375.0

3,202.5

4,160.0 2,245.0

1 ,764.75

2,284.5 1, 245. 0

1,71 9.75

1,783.5 1, 659.0

3,484.5

4, 068 .0 2, SO LO

141.18

140.15 143. 10

88.08

70.49 120.44

108.8 1

97.79 129.2 2

-+ -+ +

+

+ +

41. 18

40. 15 4 3.10

11.92

29.5 1 20.44

8.81

2.2 1 29. 22

++

++

+

+++

+

00 .....

rn

~

o ..., '"0 =-oen '0 =o..,

§

~.

~

'" 0-

::;

~

~

...,

::;

10

(1)

;>;"

10

~

c:

::;

o

~

" $" ~ §

10

I:l:i

.".

t.".l

8l " o...,

KH23~PO.

O/R

4sCa,Cl z

Over-ground parts (0)

Roots (R)

OIR

Overground parts (0)

Roots (R)

Part of the plant

Labelled compound

+ I(x) + I(x) + I(x)

+I (x)

+ I(x)

C

+I (x)

Control plants (C) Infected plants (I)

C

Control plants (C) Infected plants (I)

C

Control plants (C) Infected plants (I)

C

Control plants (C) Infected plants (I)

C

Control plants (C) Infected plants (I)

C

Control plants (C) Infected plants (I)

State of the plant

351,163.0

417,872.5 284,453.5

1808,070.5

1913,529.5 1702,611.5

0.2008

0.2330 0.1686

149,599.5

159,816.5 139,382.5

74,801.5

55,317.5 94,285.5

2.242425

3.0050 1.47985

Cultivar Kasticka x (= 100)

0.421225

0.5299 0.31255

170,497.5

195,729.0 145,266.0

103,050.0

99,250.0 106,850.0

1.8282

2.2332 1.4232

497,256.0

484,033.5 510,478.5

1291,486.75

946,326.5 1636,647.0

x

Cutlivar Hodoninka

141.60

ll5.83 179.46

71.43

49.45 96.13

209.77

227.42 185.38

113.97

122.47 104. 22

137.76

179.42 113.33

81.53

74.32 96.17

%

13.97

22.47 4.22

37.76

79.42 13.33

+

+

+

+ + +

41.60

15.83 79.46

28.57

50.55 3.87

++ + ++ +

++

++++

+ 127.42 + + + 85.38 + 109.77 + + +

+ + +

+ + +

18.47

25.68 3.83

% ± of Kasticka

Table 5. Specific activity of roots and overground parts of control and infected plants of alfalfa in counts/min./g. of dry weight (x). Differences expressed as per cents of the cultivar Hodoninka as compared to the cultivar Kasticka

~

:::

:>:i

0::

H

;.-

>-3

r-'

t--:l

00

~

Explanations:

Na 230SO(

+= ++ +++ + +++

s ignificance at

Overground parts (0)

Roots (R)

OIR

IX

+ I(x) + I(x)

=

C (x)

0.25 0.10 0.05 0.025

+I

Control plants (C) Infected plants (I)

C

Control plants (C) Infected plants (I)

C

Control plants (C) Infected plants (I)

138,384.0

178,068.5 98,699.5

118,777.0 77,624.5 98,200.75

379,251.5

303,245.5 455,257.5

0.42565

0.620S 0.2305

259,828.75

256,074.0 263,583.5

0.3940

0.4712 0.316S

140.92

149.92 127.15

145.96

l1S.42 172.72

10S.03

131. 75 72.76

+ + +

+ + + +

+

40.92

49.92 27.15

45.96

18.42 72.72

8.03

31.75 27.24

+

+

+ +

~

IX

00

.:: 00

g

5 ].

'"d

o ......

::l

c·

.,..

C> ID

o

1'2..

iil:::

f"'l

~

::l

P>

ct>

It ;0;-

~

~

o

~ ::;:-

~

:!.

?t

t:ti ~

a,

C> .,..

~

t;j

384

L.

TAIMR

u. a.

cultivar Kasticka. The radioactivity of 32p per plant was also significantly higher by 39.01 % (IX = 0.05) in infected Hodoninka plants, as compared to Kasticka (Table 4). A comparison of the specific activities of the overground organs and roots, expressed in terms of 0/R values, reveals the following differences between both investigated cultivars (Table 5): For 32P, healthy plants of the cultivar Hodoninka showed significantly higher 0/R values in comparison with Kasticka (by 127.42 %, IX = 0.10). This proves that the 32p transport from the roots to the overground parts is much more intensive in Hodoninka. For 35S, the 0/R value in Hodoninka was non-significantly higher (by 31.75 %) than in Kasticka. Infected plants of the cultivar Hodoninka showed non-significantly higher 0/R values for 32p (by 85.38 %) and non-significantly lower ones for 35S (by 27.24%), as compared with Kasticka. The most conspicuous difference between both cultivars was found in the 45Ca uptake and translocation. Plants of the resistant cult ivaI' Hodoninka contained 55.82 % more 45Ca by healthy plant than the susceptible cultivar Kasticka. This difference is significant (IX = 0.05) (Table 4). After the infection, the 45Ca content decreased in the resistant cultivar, while it increased in the susceptible one. Nevertheless, the 45Ca content remained non-significantly higher (by 15.41 %) in the infected resistant cultivar, as compared with the susceptible one (Table 4). Healthy plants of the cultivar Hodoninka showed a non-significantly lower (by 25.68%) 0/R value than those of the cultivar Kasticka, which means that the 45Ca transport from the roots to the overground organs was relatively more intensive in Kasticka than in Hodoninka. In the infected plants of both the cultivars the 0/R values were about identical (in Hodoninka they were non-significantly lower by 3.83 %. Table 5). A comparison of the average amount of 45Ca per plant in the overground organs of both cultivars shows healthy Hodoninka plants to contain significantly (IX = 0.025) more 45Ca (by 51.89 %) than plants of the cult ivaI' Kasticka. However, following the infection, the 45Ca content of the overground organs attained almost the same level in both cult ivaI's (it remained non-significantly higher by only 2.80 ~o in Hodoninka) (Table 4).

Discussion From the average values of the specific activities, established for the single radioisotopes, it follows that after the infection the content of labelled elements was always higher in the roots, while it decreased in the overground organs. This might be considered to provide indirect evidence for the assumption that bacterial wilt of alfalfa does not primarily damage the water potential of the root cells, thus limiting the uptake of water and nutrients into the plants. It seems more probable that a dysfunction of the vascular system, limiting or blocking the transport of water and nutrients to the overground organs, is more important for the development of the syndrome of the disease. It is assumed that the absence of radioisotopes, observed in the autoradiograms at first on the margins of the leaves and in more advanced stages of the disease also in the stalks, confirms the view that the high molecular weight polysaccharides, blocking together with the bacterial mass, the xylem tubes may play an important role in bacterial wilt pathogenesis. HODGSON, PETERSON, and RIKER (1949) have shown that polysaccharides in the tracheae (galactose and fucose produced by Corynebacterium insidiosum may be involved in bacterial witl of alfalfa, SPENCER and GORIN [1961]; RIES and STROBEL [1970]) increase the viscosity of the xylem flow or

Effect of Bacterial Wilt on Uptake and Translocation of Phosphorus

385

entirely block at first the narrowest parts of the veins, ending on the margins of the leaf blade, and later they close the vessels on the top and, eventually, on the base of the stalk. SUTTON and WILLIAMS (1968) report that the polysaccharides, present in the veins of detached cabbage leaves, infected by Xanthomonas campestris, block the 32PO4" 1 and eosin transport. Bearing in mind the fact that the phosphorus, sulphur, and manganese uptake by alfalfa plants and the transport of these elements to the overground organs was markedly reduced, owing to a dysfunction of the xylem vessels, how is it possible to explain that Ca uptake by infected plants of the susceptible cultivar was the same as in hEalthy plants and that an accumulation took place mainly in the overground organs? It might be assumed that translocation of Ca, taken up by the roots of alfalfa, does not occur via the transpiration stream in the xymel vessels. Taking into account other known possible pathways of transport (via the phloem, the apoplast or the intercellular spaces) the Ca translocation via the apoplast (the cell walls) seems most probable. CRAFTS and YAMAGUCHI (19()0) report that the 45Ca, applied to the bases of Phaseolus laeves, moved uniformly through the leaf blade from the site of application, but did not appear in the vascular bundles of the leaf blade, the petiole or the stalk, as observed with labelled 2,4-dichlorophenoxy acid. According to some authors the transport of calcium compounds might occur also through the phloem, but the velocity of their translocation is negligible (RUBIN 1963). In view of the marked differences in the calcium uptake and translocation prior to and following the infection, the role of calcium in the defence mechanism of alfalfa against Corynebacterium insidiosum was taken into consideration. Healthy plants of the resistant cultivar Hodoninka contained significantly more Ca per plant than plants of the susceptile cultivar Kasticka. It is surprising that after the infection a decrease was observed in the resistant cultivar, while even a slight increase occurred in the susceptible one. It is difficult to explain this phenomenon otherwise than by the assumption that calcium was leaking from the plants to the nutrient medium in infected plants of the resistant cultivar. This process might possibly enable the resistant plants to take up another cation in exchange for calcium. The specific activity of calcium decreased in the overground organs and increased in the roots as a result of infection. In healthy plants the specific activity was 2.23 to 3.00 times higher in the overground organs than in the roots, while following infection it was only 1.42 to 1.47 times higher. It is noteworthy that the overground organs of healthy plants of the resistant cultivar contained a significantly larger amount of 45Ca than those of the susceptible one. However, after the infection the Ca-content of the overground organs was about equal in both cultivars. In the Fusarium wilt of cotton, caused by Fusarium oxysporum f. vasinJect~tm, SARASWATHI-DEVI and SADASIVAN (1961), using spectral analysis, showed leaves of susceptible plants to contain a larger amount of ionized calcium as compared to resistant ones, while the opposite was true with healthy plants. The fact that the Eame changes in Ca-content were ascertained in cotton infected by Fusarium vasinfectum as in alfalfa infected by Corynebacterium insidiosum favours the view that calcium plays a more general role in the pathogenesis of bacterial wilt. From the literature concerning the role of calcium in resistance of the plants to pathogens of bacterial wilt, the paper of EDGINGTON and WALKER (1958) may be cited, pointing to an increase in susceptibility of tomato plants to Fusarium wilt, brought about by Ca-deficiency. This is obviously connected with the higher resistance of pectin substances of the host to hydrolysis by pectolytic enzymes secreted by the pathogen (EDGINGTON, CORDEN, and DIMOND 1961). SARASWATHI-DEVI and SADASIVAN (19{)l) explained accumulation of ionized calcium in cotton leaves of infected susceptible plants in two fashions: 1. calcium is Zbl. Bakt. II. Abt., Bd. 130

386

L. TAlMR u a., Effect of Bacterial Wilt on

transported to the overground organs by the transpiration stream as a complex toxin-Ca (fusaric acid-Ca). In the leaves this complex dissociates, and Ca is accumulated in the form of ions. 2. P ectolytic enzymes of the pathogen release calcium from the calcium pectates, and the element is transported to the leaves by the transpiration stream. None of the adduced explanations seems to by congenial for alfalfa infected by bacterial wilt. Since the pathogen of bacterial wilt does not produce fusaric acid or a similar toxic compound, it is possible to rule out the assumption that Ca accumulates in the overground organs of susceptible plants as a result of diSintegration of a toxinCa complex. In consideration of our present results the following suggestion is proposed: A relatively higher Ca accumulation took place in the overground organs of infected plants of the susceptible alfalfa cultivar as compared to the resistant one , because in resistant plants a more intensive redistribution of calcium from the overground organs to the roots and even from the roots to the nutrient medium took place as a result of infection. Ackn o wl e dgement The authors are indebted to Ing. JOSEF MACHEK CSc., Fa,culty of Physics and Mathematics, Charles U niversity, Pra,gue , for va luable advices concerning th e statistical evaluation of the results.

References CRAFTS, A. S., and YAMAGUC HI , S.: Absorption of herbicides by roots. Am . J . Botany 47 (1960), 248 - 255. E DGINGTON, L. V., CORDEN, M. E. , a nd DIMOND, A. E.: The role of pectic substances in chemically induced resistance to Fusarium wilt of tomato. Phytopathology 51 (1961),179-182. - and WALKER, J. C.: Influence of calcium and boron nutrition on development of Fusarium wilt of to mato. Phytopathology 48 (1958),324-326. HODGSON, R., PETERSON, W. H., and R IKER, A. J.: The toxicity of p olysaccharides a,nd large molecules t o tomato cuttings. Phyto pathology 39 (1949), 37 - 62. K REITLOW, K. W.: Infection seven-day-old alfalfa seedlings with bacteria through wounded cotyledons. Phytopathology 53 (1963) , 800 - 803. RIES, S. M., and STROBEL , G. A. : Properties of high molecular weigh t t oxin produced by Corynebacterium insidio8um. Phytopa thology 60 (1970) , 1309 - 1310. R UBIN, B. A.: Kurs fiziologii . Moskva 1963. SADASIVAN, T. S.: Physiology of wilting plan ts. In : Biochemische Problcm e der Pflanze. Tagungsberich te Nr. 74, DAL Berlin 1965 , 147-163. SARASWATHI.DEVI, L., and SADASIVAN, T. S.: Cotton wilt and calcium a ccumulation. Experientia 17 (1961), 554~555. SPENCER, J. F. T., and GORIN, P . A. J.: The occurrence in the h ost plant of physiologically active gum s produced by Corynebacterium insidiosum and Corynebacteriltm sep edonicum. Can. J. Microbi oI. 7 (1961), 185-188. SUTTON, J . C. , and WILLIAMS, P. H .: X ylem plugging and black lesion development in cabba,ge. Phytopathology 58 (1968), 1069. Author 's a dd ress : lng. LUBOMiR TAIMR, esc. , Ing . ANNA K UDELOVA , Ing. VACLAV K 1JDELA, e s c. , and prom. chern . E VA BERGMANNOVA, Research Institutes for Plant Production , Insti tute of Plant Protection , 16106 P rague 6-Ruzyne (CSSR).