leakage of electrolytes and amino acids from susceptible

日植病報 45: 625-634 (1979)

Ann. Phytopath. Soc. Japan 45: 625-634 (1979)

Leakage of Electrolytes and Amino Acids from

Susceptible and Resistant Citrus Leaf Tissues

Infected by Xanthomonas citri

Macao GOTO*, Ikuko TAKEMURA* and Katsuji YAMANAKA*

後藤正夫＊・竹村育子＊・山中勝司＊: Xanthomonas citriに感染したカンキツ

葉組織からの電解質およびアミノ酸の漏出

Abstract

Conductivity of intercellular fluid sharply increased in 24 to 48hr after susceptible

Natsudaidai leaves were inoculated with Xanthomonas citri. At the same time, amino

acids were released into intercellular spaces increasing in concentration several times

in 48hr compared with uninoculated leaves. The major amino acids in intercellular

fluid from inoculated leaves were: proline, anserine, ƒÁ-amino-n-butyric acid, serine,

alanine and aspartic acid. Carnosine and ethanolamine were detected as new amino

acids in the infected tissues. Electrolyte leakage also occurred in leaves of Calamondin,

a resistant cultivar, although the growth of X. citri was kept low during the period

of observation. Strain V2 of Erwinia herbicola induced rapid leakage of electrolytes

and extremely rapid movement of amino acids into intercellular spaces in Natsudaidai

leaves. Strain F1 of the same bacterium, however, did not induce these reactions.

The bacterial populations quickly declined when incompatible xanthomonads were

inoculated into Natsudaidai leaves. There was considerable alteration in amino acid

compositions of host cells inoculated with X. phaseoli but not in those of intercellular

fluid, indicating that the host cell membrane did not suffer serious damage. The

changes found in the leaf tissues inoculated with X. citri seemed to be situated

between these two.

(Received August 13, 1979)

Introduction

In citrus leaves inoculated with Xanthomonas citri nutrient substances were released from the host cells into intercellular spaces 6 to 9hr after inoculation9). In this stage, growth of the pathogen was still in lag phase and macroscopic damage was rarely observed in the infected host tissues. These observations indicated that the host cell membrane might be damaged in the early stage of infection resulting in leakage of amino acids and/or electrolytes. This process has often been reported as typical of the host-parasite relationship in bacterial plant diseases1,5,11).

The objective of this research was to study the host-parasite interaction in citrus canker with regard to the permeability change of host cell membrane in the compat-ible and incompatible combinations, and to the histopathological changes of the infected tissues.

*Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422, Japan静岡大学農学部

Supported in part by Research Grant No.248047 from the Ministry of Education, Science

and Culture, Japan.

626 日本植物病理学会報第45巻第5号昭和54年12月

Materials and Methods

Bacteria. The bacteria used in the study included strains of X. citri, X. begoniae,

X. campestris, X. geranii, X. oryzae, X. phaseoli, X. phormicola, X. physalidicola, X.

pisi, X. pruni, X. vitians and two strains of Erwinia herbicola, respectively. All

bacteria were obtained from the culture collection of phytopathogenic bacteria at the

Laboratory of Plant Pathology, Shizuoka University. Strain U9-1 which was mainly

used for the studies with X. citri was isolated from Unshu (Citrus Unshu Marcov.)

in 1960; it is a mannitol-negative strain. Two strains, F1 and V2 of E. herbicola

were isolated from the surface of citrus leaves.

Plants. One-year-old seedlings of Natsudaidai (Citrus Natsudaidai Hayata)

and Calamondin (Citrus madurensis Loua.) were grown in a greenhouse and their fully

expanded leaves were used for inoculation. Natsudaidai was highly susceptible to

citrus canker,whereas Calamondin was resistant.

Inoculation method. A leaf infiltration technique was used in the inoculations.

Bacterial cells grown on peptone-sucrose agar slants at 28C for 24hr were suspended

in sterile distilled water, centrifuged, and resuspended in water. Turbidity of the

suspension was adjusted nephelometrically to the concentration of 2•~108cells/ml.

Heat-killed cells were prepared by boiling the bacterial suspension in water for 10

minutes.

Permeability change of host cell membrane. Electrolyte leakage was deter-

mined by conductivity changes that were measured by a conductivity meter (Model

CM-2A of TOA Electric Co. Tokyo). Leaf samples were washed with tap water,

rinsed with distilled water, and then water droplets on the leaf surface were blotted

with clean cheese cloth. Thirty disks, 0.785cm2 in size, were taken from the leaves,

placed in 50ml distilled water and incubated for 1hr on a reciprocal shaker (frequency:

117/min). Conductivity of the water was measured before and after the incubation.

Before the conductivity measurement, the water was filtered through millipore films

(0.22ƒÊm). These were previously washed thoroughly with distilled water and this

was shown not to have an effect on the conductivity of the filtrates.

Assay of ions, amino acids and carbohydrates. After conductivity was meas-

ured, water was concentrated by vacuum evaporation and subjected to flame analysis

for K+. For amino acid analysis, intercellular fluid was collected by the infiltration

technique described previously9), and filtered through millipore films. The fluids

were then assayed by a Hitachi amino acid autoanalyser Type 835. Total sugar

contents were determined by the anthrone method2).

Number of bacterial cells in leaf tissues. Surface of leaf disks was sterilized

with 0.5% hypochlorite solution, washed three times with sterile distilled water and

crushed in 10ml of 1% peptone-sucrose broth. The number of bacteria in the

peptone broth was determined by conventional dilution plating methods.

Histopathological observation of inoculated leaves. Leaf tissues were fixed

with a mixture of f ormalin, acetic acid and alcohol, dehydrated, and embedded in par-

affin by the conventional method. The embedded tissues were sectioned by a micro-

tome at a setting of 10 to 15ƒÊm.

Results

Permeability change in Natsudaidai and Calamondin leaves inoculated with

X. citri

In susceptible Natsudaidai leaves inoculated with living cells of X. citri, electrolyte

Ann. Phytopath. Soc. Japan 45 (5). December, 1979 627

Fig. 1. Changes of bacterial population in Natsudaidai

leaves inoculated with X. citri and electrolyte loss

from the leaf tissues.

•›-•› Population of X. citri; •¢-•¢ Electrolyte

loss from the leaves inoculated with living cells.

•£-•£ Electrolyte loss from the leaves infiltrated

with heat-killed cells.

Fig. 2. Changes of bacterial population in Calamondin leaves

inoculated with X. citri at the conc. of 108cells/ml and

electrolyte loss from the leaf tissues. •›-•› Population

of X. citri; •¢-•¢ Electrolyte loss from the leaves in-

oculated with living cells; •£-•£ Electrolyte loss from

the leaves infiltrated with heat-killed cells.

leakage was detected 2 days after inoculation when bacterial popula-tion reached the level of 107cells/disk (Fig. 1). The conductivity continued to increase during the six-day observation. No conduc-tivity increase was observed in the leaves infiltrated with the heat-killed cells. On the leaves inoculated with living cells, the first symptom developed 2 days after inoculation as several water-soaked and dark green dots. The dots quickly increased in number and covered whole leaf blades in 4 days. After 6 days, the tis-sues became swollen with a blister-like appearance. No pathological change occurred in the leaves infil-trated with heat-killed bacteria.

In resistant Calamondin leaves inoculated with living cells, the conductivity increase occurred in the second day. The bacterial

population still remained at the level of 105cells/disk and continued to increase until the end of observation (Fig. 2). Bacterial population reached a peak of 106cells/disk, four days after inoculation, decl-

ing afterward. There was no detectable change in the con-ductivity of the leaves infil-trated with heat-killed cells. On the fifth day, a small number of water soaked, dark

green dots appeared along the midrib on the undersur-face of leaves inoculated with living cells. The number of lesions were less than 10

spots per leaf at the inoculum dose used. These dots became dark brown necrotic spots several days after inoculation and did not develop into typical canker lesions. The whole leaves turned yellowish green in a week.

Permeability change in Natsudaidai leaves inoculated with X. phaseoli and E. herbicolaTen different xanthomonads other than X. citri were inoculated in Natsudaidai

leaves by the infiltration technique using inocula with 108cells/ml. Marked decreases in the population were observed irrespective of the bacteria used (Table 1). No symp-


Table 1. Population changes of xanthomonads in Natsu-

daidai leaves inoculated by infiltration technique


leaves inoculated with X. phaseoli at the conc. of

108cells/ml and electrolyte loss from the leaf

tissues. •›-•› Population of X. phaseoli; •¢-•¢

Electrolyte loss from the leaves inoculated with

living cells; •£-•£ Electrolyte loss from the leaves

infiltrated with heat-killed cells.


leaves inoculated with two isolates V2 and F1 of E.

herbicola. •›-•› Population of isolate V2; •œ-•œ

Population of isolate F1; •¢-•¢ Electrolyte loss

from the leaves inoculated with V2; •£-•£ Electro-

lyte loss from the leaves infiltrated with F1.

toms developed on these leaves.This experiment was repeated

with a culture of X. phaseoli (Fig. 3). The bacterial population show-ed a rapid decline and no living cells were detected 4 days after in-oculation. No conductivity change was observed. No symptom was visible on the inoculated leaves after 2 weeks.

Bacterial cells of the isolates V2 and F1 of E. herbicola were inoculated in Natsudaidai leaves following the procedure described

previously. Isolate V2 induced the hypersensitive reaction about 24hr after inoculation with devel-opment of confluent, dehydrated necrotic lesions, but isolate F1 did not elicit this response (Fig. 4). The number of cells of strain V2 as well as the conductivity in-creased at a constant rate during the 48hr observation period, whereas no comparable increases were noted with strain F1.

Histopathological changes

In the susceptible Natsudaidai

leaves inoculated with X. citri,

plasmolysis was observed in a

small number of parenchyma cells

2 days after inoculation. After 3

days, these plasmolysed cells in-

creased in number and bacterial

colonies were stained. In 4 days

after inoculation, masses of bacte-

rial cells and slime occupied some

parts of the intercellular spaces.

Typical, hypertrophied cells were

not observed in the tissues 7

days after inoculation. In the

resistant Calamondin leaves that

were inoculated with living X. citri

cells, bacterial strands composed

of bacterial cells and slime were

not detected in the intercellular

spaces until the end of the obser-

vation period. The sole patho-

logical change was the develop-

ment of small number of plasmo-


lysed and necrotic cells around the vascular bundles. These cells were characterized

by deep staining in contrast to the other cells in the same regions. The necrotic

cells were detected in small number in the sections as early as 24hr after inoculation.

They increased in number with passage of time but were always distributed around

the veins. The necrotic cells could not be detected in the leaf tissues that were

infiltrated with heat-killed bacteria.

Analysis of the diffused substances from leaf disks

The concentration of K+ and total amino acids increased in parallel with the

Fig. 5. Release of K+, total amino acids and total sugars from

the Natsudaidai leaves inoculated with X. citri at the

conc. of 108cells/ml.

•›-•› •œ-•œ K+ content; • -• •¡-•¡ Total amino

acid content; •¢-•¢ •£-•£ Total sugar content. Open

•c From the leaves inoculated with living cells; Closed

From the leaves infiltrated with heat-killed cells.

conductivity (Fig. 5). Change

of total sugar content was

different in pattern from

those of the ions and amino

acids. This may be attribut-

able to the polysaccharides

produced by the pathogen.

The total of amino acids was

assayed by the ninhydrin

reaction. For amino acid

analysis, the intercellular flu-

id was taken from Natsudai-

dai leaves inoculated with 2

•~ 108cells/ml of X. citri, X.

phaseoli and E. herbicola,

respectively. The intercellu-

lar fluid taken from leaves

infiltrated with sterilized dis-

tilled water was used as the

control. The inoculated and

non-inoculated leaves showed

no difference in dry weight

at the time of sampling.

Intercellular fluid was taken

24 and 72 hr after inoculation

with X. citri, 48hr with X. phaseoli, and 12hr with E. herbicola.

Intercellular fluid of healthy leaves contained Pro, Ans (ƒÀ-Alanyl-methyl-histidine),

Ser and GABA (ƒÁ-Amino-n-butylic acid) in order of concentration followed by Asp,

Ala, Hy-Lys and Glu (Table 2 and 3). In the leaves infected by X. citri, the amino

acids such as Gly, Arg, Ala, GABA, Cys, Thr, Pro, Leu and Lys increased markedly.

Concentrations of these amino acids increased to levels more than 3 times higher than

the concentrations in healthy leaves. The increase was observed also in other amino

acids with the exception of p-Ser and Glu which decreased significantly. In the

homogenates of the citrus leaves prepared after intercellular fluid was removed, Pro,

Asp, GABA, Ans and Ser were the major amino acids followed by Ala, Hy-Pro, Eta-

NH2 and Lys. When infected by X. citri, however, Met, Phe and His showed con-

centrations about 3 times higher, whereas the other amino acids increased 1.2 to 2.4

times. In contrast, appreciable increases were not observed with Ans, Glu, Pro, Ser

and Asp. Gly, Cysthi, Eta-NH2, Try and ƒÀ-Ala were detected only in the intercellu-

lar fluid taken from the inoculated leaves. The above changes in concentration were

parallel to those in the homogenates of leaf tissues.

Intercellular fluid from Natsudaidai leaves inoculated with X. phaseoli contained


Table 2. Amount of amino acids (ƒÊ mole/ml) in intercellular fluid of Natsudaidai

leaves inoculated with X. citri, X. phaseoli and E. herbicola at different

time intervals after inoculation1)

1) Intercellular fluid was directly loaded on Hitachi amino acid autoanalyser Type 835.2) Hours after inoculation.

Cys 24 times higher than in control samples, whereas the other amino acids decreased significantly or did not change. In contrast to the intercellular fluid several amino acids such as Glu, Ala, Phe, Lys and His increased greatly in the leaf homogenates. In general, the changes in the homogenates were similar in magnitude to those in the leaves infected by X. citri; in contrast changes in intercellular fluid were signifi-cantly less than those of the homogenates.

In comparison with the leaves inoculated with X. citri and X. phaseoli, the amount of amino acids in the intercellular fluid was greatly changed in leaves inoculated with E. herbicola, isolate V2. In the leaves 24hr after inoculation, Cys and Arg increased 100 times above concentrations in healthy leaves. These were followed in order by Tyr, Phe, GABA, Ans, Ala, Gly, Thr, Pro, Lys and Val which increased 10 to 70 times. Although the increase was noticed within 12hr after inoculation, the major


Table 3. Amount of free amino acids in the homogenates of the residual leaf tissues of Natsudaidai after intercellular fluids were collected1)

1) After intercellular fluid was removed, 5g leaves were homogenized in 50ml of distilled water and centrifuged. The precipitate was again treated in the same way as the above.The supernatants were combined and evaporated under vacuum to 5ml. Sulfosalicylic acid was added to it at the conc. of 3% to remove proteins. The mixture was centrifuged and the supernatant was assayed.

2) Hours after inoculation.

increase occurred after 24hr. In contrast, Ser, Asp and Ans increased slightly or even decreased. The homogenates of residual leaf tissues were characterized by

pronounced decrease in many amino acids. Some amino acids such as Gly, Tyr, Phe and Arg increased only 2 to 6 times over the levels in healthy leaves.

Discussion

In the susceptible Natsudaidai leaves inoculated with X. citri, K+ and amino acids were released into intercellular spaces in parallel with the rise of conductivity. The


increase occurred when the bacterial population increased above 107cells/cm2 and the

symptoms developed subsequently. In the resistant Calamondin leaves, however,

the conductivity rose when bacterial population was below 106cells/cm2 and no symp-

toms appeared except for a few tiny necrotic spots. The former pattern was analo-

gous to bacterial leaf spot of cucumber caused by Pseudomonas lachrymans in which

development of water-soaked lesions coincided with increase of conductivity, amino

acids, reducing sugar and proteins15). These phenomena indicated that X. citri

inoculated to citrus leaves induced permeability changes of the host cell membranes

and caused leakage of nutrient substances into the intercellular spaces. As has been

reported in a previous paper9), it is likely that amino acids started to be released

within 12hr after inoculation. The amino acids or electrolytes that leaked out from

the leaf cells in the early stages, however, could be small in amount because of the

less severe effects on the host cells, and therefore these were diluted in the water

suspending the leaf disks.

In contrast to Natsudaidai, the resistant Calamondin leaves showed electrolyte

leakage when bacterial populations were relatively low. This conductivity increase

could be explained on a different basis from that applicable in the case of Natsudaidai

leaves. It is likely that hypersensitivity reactions are involved in this case. Similar

phenomena were observed in the incompatible combination in the leaves of pepper or

tobacco in which conductivity increase without bacterial multiplication4,7,8,11,12). How-

ever, histopathological observation proved that typical hypersensitivity reactions are

not induced in citrus leaves. In the combination of resistant Calamondin and X. citri,

the necrosis occurred in only a limited number of cells surrounding vascular bundles,

but never in the areas of leaf blades. Bacterial cells were never surrounded by killed

host cells as was observed in typical hypersensitivity reactions in tobacco and pepper

leaves. Although the same doses of inoculum were used as in Natsudaidai leaves,

the bacterial population in Calamondin leaves were 1/100th of those in Natsudaidai

throughout the period of experiments. This fact indicated that toxic substances such

as phytoalexins were released from host cells10). In Natsudaidai leaves inoculated

with X. phaseoli, the bacterial population also quickly declined without an increase

of conductivity of any histopathological changes in the leaf tissues. The differen-

tially toxic substances occasionally found in intercellular fluid of healthy citrus leaves

may have important roles in the early stage of host-parasite interaction in citrus

canker10).

The histopathological observation of citrus leaves inoculated with X. citri by the

infiltration technique indicated differences from those by stomatal inoculation or wound

inoculation in that the typical hypertrophied cells scarcely observed. This fact

indicated that the amount and/or activity of auxins produced either by bacterium or

host cells at the site of interaction were quite different depending on the inoculation

method.

Distinct contrasts were demonstrated between X. phaseoli and E. herbicola in the

number of amino acids showing the appreciable increase or decrease in the inoculated

leaves. In the former, amino acid concentration in the host tissues increased consid-

erably but these amino acids were not released into intercellular fluid. In the leaves

inoculated with E. herbicola, on the contrary, amino acids in the host tissues showed

considerable decrease, whereas those in the intercellular fluid increased greatly. The

situation was intermediate in the leaves inoculated with X. citri where amino acid

concentrations increased either in intercellular fluid or host tissues in moderate degree.

Several amino acids such as ƒÀ-Ala, Cysthi, Eta-NH2, Try and Car (ƒÀ-Alanyl-histidine)

were detected as new amino acids in the intercellular fluid from the inoculated


leaves. The number and/or kinds of the major amino acids above 0.1ƒÊ mole/ml

sample, found in intercellular fluid and leaf tissues were in the similar trends as the

intensity of the changes mentioned above. Pro was consistently highest in concen-

tration regardless of inoculation, and GABA increased irrespective of the organisms

inoculated. In the leaves inoculated with X. citri, amount of Ala and Asp increased

in intercellular fluid, and Arg, Car and Ala in the host cells. Increase of Arg was

characteristically noticed in these host tissues in which considerable leakage of amino

acids had occurred.

The amino acid composition of intercellular fluid and host tissues as well as their

alteration after inoculation indicated that the pyruvate and/or ƒ¿-keto-glutarate path-

ways of amino acid metabolism were predominantly activated. It was interesting

that the dipeptide Ans (ƒÀ-Alanyl-methyl-histidine) was consistently detected as one

of the major amino acids in the leaves and an another dipeptide Car (ƒÀ-Alanyl-histidine)

was detected in the tissues infected with X. citri or E. herbicola. These two

dipeptides have been detected in animal cells13). Eta-NH2 is a composition of phospho-

glycerids so that its detection in the inoculated tissues indicated the decomposion of

host cell membrane. The very rapid increase of amino acids and quick rise of

conductivity indicated that E. herbicola induced serious dysfunction of the host cell

membrane which led to the hypersensitivity reaction. In contrast, the tissues inocu-

lated with X. citri seemed to induce mild alteration of membrane permeability so that

accumulation of several amino acids supported the continuous growth of X. citri in

the early phases of infection. When infected by X. phaseoli, no appreciable change

was assumed in host cell membrane because no change of conductivity and amino

acid concentrations were detected, although the amino acid metabolism was evidently

altered in host cells.

Literature cited

1. Addy, S.K. (1976). Phytopathology 66: 1403-1405.

2. Ando, E., H. Terayama, K. Nishizawa and T. Yamakawa (1967). Methods for Biochemistry

I, II (in Japanese). Asakura Shoten, Tokyo. pp. 822.

3. Basham, H.G. and D.F. Bateman (1975). Phytopathology 65: 141-153.

4. Cook, A.A. and R.E. Stall (1968). Phytopathology 58: 617-619.

5. Cook, A.A. and R.E. Stall (1977). Phytopathology 67: 1101-1103.

6. Gardner, J.M .and C.I. Kado (1976). Jour. Bact. 127: 451-460.

7. Goodman, R.N. (1965). Phytopathology 55: 217-221.

8. Goodman, R.N. (1968). Phytopathology 58: 872-873.

9. Goto, M., Y. Tadauchi and N. Okabe (1979). Ann. Phytopath. Soc. Japan 45: 618-624.

10. Goto, M. unpublished data

11. Klement, Z. and R.N. Goodman (1967). Ann. Rev. Phytopath. 5: 17-44.

12. Klement, Z. (1972). Plant Pathogenic Bacteria 1971, Proc. III Internat'l Conf. Plant Path.

Bact., Wageningen, 14-21 April 1971. p. 157-164.

13. Lehninger, A.L. (1975) Biochemistry, 2nd ed. Worth Publishers, Inc. New York. p. 96, 672.

14. Tseng, T.C. and M.S. Mount (1974). Phytopathology 64: 229-236.

15. Williams, P.H. and N.T. Keen (1967). Phytopathology 57: 1378-1385.


和文摘要

Xanthomonas citri に感染したカンキツ葉組織からの電解質

およびアミノ酸の漏出

後藤正夫・竹村育子・山中勝司

X. citriを接種したナツミカン葉組織では病菌増殖に伴って24～48時間目から電解質およびアミノ酸の漏

出が認められた。主なアミノ酸の種類はプロリン,アンセリン,γ-アミノ酪酸,セリン,アラニン,アスパ

ラギン酸,カルノシンおよびエタノールアミン等で,カルノシンとエタノールアミンは感染組織のみに検出

された。抵抗性のカラモンジンでは細菌増殖は低く抑えられたが電解質漏出は起った。Erwinia herbicola

のV2菌株では漏出が特に急激に起ったが,同F2菌株では認められなかった。X. phaseoliでは電解質およ

びアミノ酸の漏出は起らなかったが,細胞のアミノ酸組成には大きな変化が認められた。X. citriを接種し

たナツミカン葉の変化はこれらE. herbicola V2菌株とX. phaseoliのほぼ中間的なものであった。

leakage of electrolytes and amino acids from susceptible

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