- Email: [email protected]
β-1,3–Glucanase in The Rhizosphere and on Plant Roots
6-1,3-GLUCANASE IN THE RHIZOSPHERE AND ON PLANT ROOTS Sotolova I., Jandera A., Hanzlikovd A. Institute of Microbiology, Czechoslovak Academy of Sciences, 142 20 Prague 4, Czechoslovakia
ABSTRACT Air-dried soil exhibited no ~-1,3-glucanaseactivity; the enzyme in produced by wetting the soil and enriching it with glucose or casein hydrolysate. Addition of @-1,3-glucan to the soil led to a further increase in the activity which did not take place in the presence of glucose. @-1,3-glucanase on barley root surface is mostly produced by the plant, with a negligible contribution from seed microflora. The enzyme is bound to the root surface structures and its activity depends on the barley variety and the time of germination. The activity on the root surface is 1000-fold higher than in the rhizosphere soil which is on ave,rageonly 10-50 fold higher than that in free soil depending on barley variety and soil type. Addition of glucan to soil with germinating barley plants induces a marked increase in the activity of the enzyme in rhizosphere soil while the activity on plant roots remains unchanged. We isolated a Gram-positive bacterium producing @-1,3-glucanase in the presence of glucan in the stationary growth phase.
INTRODUCTION The study of enzymic lysis of cell walls of hyphae and fungal spores by microorganisms contributes to the understanding of the mechanism of decomposition of the walls in soil. The results obtained constitute a basis for the application of lytic enzymes in the control of some fungal soil pathogens (Mitchell and Alexander,l963; Mitche11,1963). The lytic activity is characteristic of microorganisms producing 1,3- 8-glucanases and chitinase (EC 3.2.1.14). In addition, on plant roots 1,3- B-glucanases can release the so-called elicitor from cell walls of plant pathogenic microorganisms which can further stimulate the production of phytoalexins by plants thus playing an important role in the protection against pathogens (Keen and Yoshikawa,l983). In order to clarify these phenomena it is necessary to understand the activity of 1,3- 8-glucanases as well as the conditions required for their
-301-
production and the occurence of microorganisms producing these enzymes in soil and on plant roots.
MATERIALS AND METHODS Samples of chernozem soil (pH7.5;C:N 9.7:l) were taken from the surface arable layer and used in the form of air-dried structural aggregates 0.2-2.0 nun in diameter. Soil cultivation (27 'C, 60% WHC), germination and cultivation of barley, determination of C02 production in soil were described earlier (Vbgnerovb et aL.,l960, RySavq and Macura,1972). The activity of B-lf3-glucanasewas determined according to Lethbridge et al. (1978) 1.0 g soil or 0.25 g roots of barley, 2.0 ml 0.1% sodium azide, 2.0 ml 1% 8-lf3-glucan in 0.1M sodium-maleate buffer (pH 5 . 4 ) , incubation 17h at 37 'C, assayed for glucose. An isolated rhizosphere bacterium was cultivated on Taylor's medium without soil extract (Taylor,1951).
-
RESULTS AND DISCUSSION The enzyme B-1,3-glucanase is a component of root exudates of germinating barley. Its activity depends on barley variety and time of germination (Fig. 1). To prove the enzyme origin on barley roots, the plants were grown under sterile conditions. B-1,3-glucanase on barley root surface was mostly produced by the germinating plant with a negligible contribution from seed microflora. The enzyme was firmly bound to surface barley root structures and was not releasea to the clutivated medium (water). Then, the enzyme barley. roots cannot probably reduce the occurrence of fungal fytopathogens in rhizosphere soil and their possible contact with host plants. Air-dried soil did not exhibit any measurable p1,3-glucanase activity. A simple wetting of the soil resulted in the enzyme formation. A similar situation was observed when the soil was supplemented by glucose (0.6%) or by caseine hydrolyzate (0.6%). Addition of a small amount of glucan (0.2%) to the soil significantly increased the enzyme activity. No increase could be detected in the presence of glucose (Fig.2). If the soil is not supplemented with glucan, the enzyme is probably synthesized only constitutively, but if glucan is added the enzyme is produced also inducibly (SotolovA and Jandera, 1985). The course of enzyme synthesis is apparently related to the nutritional conditions under which the heterogenous microbial community grows. A possible relationship between the enzyme production and the composition of the microbial accosiation cannot be excluded.
-302-
A Q 40 -
.:
30
-
L,; -=”
Fig. 1. Activity of 8-1,3-glucanase ( A , pkat.0.259-1 roots) on the roots of germinating barley in water. a, changes of enzyme activity during germinating of barley var. Karat; b, enzyme activity on 7th day of germinating barley different varieties
1, Koral; 2, Mars; 3, Krystal; 4 , Karat; 5, Erfa Changes in glucanase activity during germination in the soil were observed on roots of barley and in the rhizosphere soil of this plant. The activity in the rhizosphere soil was always higher than in free soii, depending on the barley variety and soil type used (rhizosphere effect). The addition of glucan (0.2%) to soil with germinating barley resulted in a pronounced increase of enzyme activity in the rhizosphere soil, whereas the activity of germinating barley roots was not influenced (Table 1). The control of the enzyme activity in rhizosphere soil is probably similar as in free soil. The activity of ~-1,3-glucanasewas considerably higher on roots of barley growing in the soil than in the rhizosphere or free soil. We isolated a gram-positive bacterium from rhizosphere soil of germinating barley plant. This bacterium, inhibiting the growth of Fusarium oxysporum (Sotolovd - unpublished results from PhD thesis) produces ~-1,3-glucanasein the presence of glucan ( 0 . 2 % ) in the stationary growth phase (Fig. 3).
-303-
201
c
l'i\
Fig. 2. Total C02 production (R, mg.25 g-l soil) (top) and 1,3- 0 -glucanase activity ( A, pkat.9-l soil) (bottom) during cultivation of soil a, wetting only; b, supplemented with glucose; c) supplemented with casein hydrolyzate. Full symbols designate values obtained in soil supplemented with additional lI3-B-glucan.
-304-
Table 1. Activity of B-1,3-glucanase (pkat.g-' of dry soil or roots) during germination of barley var. Karat in soil. Glucan (0.2%) was added to the soil in the time of sowing barley Locality of enzyme
Days of germination 3
4
5
6
0.7
1.0
0.6
1.0
Rhizosphere soil
13.4
10.0
12.0
15.0
Rhizosphere soil plus glucan
26.6
22.1
14.8
15.0
1122.6
1239.3
1327.1
1280.0
802.4
1127.0
1240.0
1422.6
Free soil
Surface roots Surface roots in soil with glucan
00 IS
A
-300 10
- 200
5
0
20
10
h
Fig. 3. The growth of bacterium isolated from rhizosphere of germinating barley (1) and the activity of B-1,3-glucanase (2) of this bacterium. Glucan was added to the cultivation medium with the inoculum. OD, optical density; A , activity of enzyme, nkat
-305-
REFERENCES KEEN, N.T., YOSHIKAWA, M.: 1,3-@-glucanase from soybean releases eliactive carbohydrates from fungus celr wall. Plant Physiol. citor 71: 460-465, 1983. LETHBRIDGE, G.A., BULL, A.T., BURNS, R.G.: Assay and properties of @-1,3-glucanase in soil. Soil Biol. Biochem. 10: 389-392, 1987. MITCHELL, R., ALEXANDER, A.: Lysis of soil fungi by bacteria. Can.J.Microbiol.9: 169-175, 1963. MITCHELL, R.: Addition of fungal cell-wall components to soil for biological disease control. Phytopathology 53: 1068-1073, 1963. RYsAfl, P., MACURA, J.: The assay of 6-galactosidase in soil. Folia Microbiol. 17: 370-374, 1972. SOTOLOVA, I., JANDERA, A.: Activity of 1,3+-D-glucanase in soil. Folia iicrobiol. 30: 521-524, 1985. TAYLOR, C.B.: The nutritional requirements of the predominant flora of the soil. Proc.Soc.Appl.Bact. 14: 101-102, 1951. VAGNEROVA, K., MACURA, J., EATSKA, V.: Rhizosphere microflora of wheat. Folia Microbiol. 5: 298-310, 1960.
-
ABSTRACT Air-dried soil exhibited no ~-1,3-glucanaseactivity; the enzyme in produced by wetting the soil and enriching it with glucose or casein hydrolysate. Addition of @-1,3-glucan to the soil led to a further increase in the activity which did not take place in the presence of glucose. @-1,3-glucanase on barley root surface is mostly produced by the plant, with a negligible contribution from seed microflora. The enzyme is bound to the root surface structures and its activity depends on the barley variety and the time of germination. The activity on the root surface is 1000-fold higher than in the rhizosphere soil which is on ave,rageonly 10-50 fold higher than that in free soil depending on barley variety and soil type. Addition of glucan to soil with germinating barley plants induces a marked increase in the activity of the enzyme in rhizosphere soil while the activity on plant roots remains unchanged. We isolated a Gram-positive bacterium producing @-1,3-glucanase in the presence of glucan in the stationary growth phase.
INTRODUCTION The study of enzymic lysis of cell walls of hyphae and fungal spores by microorganisms contributes to the understanding of the mechanism of decomposition of the walls in soil. The results obtained constitute a basis for the application of lytic enzymes in the control of some fungal soil pathogens (Mitchell and Alexander,l963; Mitche11,1963). The lytic activity is characteristic of microorganisms producing 1,3- 8-glucanases and chitinase (EC 3.2.1.14). In addition, on plant roots 1,3- B-glucanases can release the so-called elicitor from cell walls of plant pathogenic microorganisms which can further stimulate the production of phytoalexins by plants thus playing an important role in the protection against pathogens (Keen and Yoshikawa,l983). In order to clarify these phenomena it is necessary to understand the activity of 1,3- 8-glucanases as well as the conditions required for their
-301-
production and the occurence of microorganisms producing these enzymes in soil and on plant roots.
MATERIALS AND METHODS Samples of chernozem soil (pH7.5;C:N 9.7:l) were taken from the surface arable layer and used in the form of air-dried structural aggregates 0.2-2.0 nun in diameter. Soil cultivation (27 'C, 60% WHC), germination and cultivation of barley, determination of C02 production in soil were described earlier (Vbgnerovb et aL.,l960, RySavq and Macura,1972). The activity of B-lf3-glucanasewas determined according to Lethbridge et al. (1978) 1.0 g soil or 0.25 g roots of barley, 2.0 ml 0.1% sodium azide, 2.0 ml 1% 8-lf3-glucan in 0.1M sodium-maleate buffer (pH 5 . 4 ) , incubation 17h at 37 'C, assayed for glucose. An isolated rhizosphere bacterium was cultivated on Taylor's medium without soil extract (Taylor,1951).
-
RESULTS AND DISCUSSION The enzyme B-1,3-glucanase is a component of root exudates of germinating barley. Its activity depends on barley variety and time of germination (Fig. 1). To prove the enzyme origin on barley roots, the plants were grown under sterile conditions. B-1,3-glucanase on barley root surface was mostly produced by the germinating plant with a negligible contribution from seed microflora. The enzyme was firmly bound to surface barley root structures and was not releasea to the clutivated medium (water). Then, the enzyme barley. roots cannot probably reduce the occurrence of fungal fytopathogens in rhizosphere soil and their possible contact with host plants. Air-dried soil did not exhibit any measurable p1,3-glucanase activity. A simple wetting of the soil resulted in the enzyme formation. A similar situation was observed when the soil was supplemented by glucose (0.6%) or by caseine hydrolyzate (0.6%). Addition of a small amount of glucan (0.2%) to the soil significantly increased the enzyme activity. No increase could be detected in the presence of glucose (Fig.2). If the soil is not supplemented with glucan, the enzyme is probably synthesized only constitutively, but if glucan is added the enzyme is produced also inducibly (SotolovA and Jandera, 1985). The course of enzyme synthesis is apparently related to the nutritional conditions under which the heterogenous microbial community grows. A possible relationship between the enzyme production and the composition of the microbial accosiation cannot be excluded.
-302-
A Q 40 -
.:
30
-
L,; -=”
Fig. 1. Activity of 8-1,3-glucanase ( A , pkat.0.259-1 roots) on the roots of germinating barley in water. a, changes of enzyme activity during germinating of barley var. Karat; b, enzyme activity on 7th day of germinating barley different varieties
1, Koral; 2, Mars; 3, Krystal; 4 , Karat; 5, Erfa Changes in glucanase activity during germination in the soil were observed on roots of barley and in the rhizosphere soil of this plant. The activity in the rhizosphere soil was always higher than in free soii, depending on the barley variety and soil type used (rhizosphere effect). The addition of glucan (0.2%) to soil with germinating barley resulted in a pronounced increase of enzyme activity in the rhizosphere soil, whereas the activity of germinating barley roots was not influenced (Table 1). The control of the enzyme activity in rhizosphere soil is probably similar as in free soil. The activity of ~-1,3-glucanasewas considerably higher on roots of barley growing in the soil than in the rhizosphere or free soil. We isolated a gram-positive bacterium from rhizosphere soil of germinating barley plant. This bacterium, inhibiting the growth of Fusarium oxysporum (Sotolovd - unpublished results from PhD thesis) produces ~-1,3-glucanasein the presence of glucan ( 0 . 2 % ) in the stationary growth phase (Fig. 3).
-303-
201
c
l'i\
Fig. 2. Total C02 production (R, mg.25 g-l soil) (top) and 1,3- 0 -glucanase activity ( A, pkat.9-l soil) (bottom) during cultivation of soil a, wetting only; b, supplemented with glucose; c) supplemented with casein hydrolyzate. Full symbols designate values obtained in soil supplemented with additional lI3-B-glucan.
-304-
Table 1. Activity of B-1,3-glucanase (pkat.g-' of dry soil or roots) during germination of barley var. Karat in soil. Glucan (0.2%) was added to the soil in the time of sowing barley Locality of enzyme
Days of germination 3
4
5
6
0.7
1.0
0.6
1.0
Rhizosphere soil
13.4
10.0
12.0
15.0
Rhizosphere soil plus glucan
26.6
22.1
14.8
15.0
1122.6
1239.3
1327.1
1280.0
802.4
1127.0
1240.0
1422.6
Free soil
Surface roots Surface roots in soil with glucan
00 IS
A
-300 10
- 200
5
0
20
10
h
Fig. 3. The growth of bacterium isolated from rhizosphere of germinating barley (1) and the activity of B-1,3-glucanase (2) of this bacterium. Glucan was added to the cultivation medium with the inoculum. OD, optical density; A , activity of enzyme, nkat
-305-
REFERENCES KEEN, N.T., YOSHIKAWA, M.: 1,3-@-glucanase from soybean releases eliactive carbohydrates from fungus celr wall. Plant Physiol. citor 71: 460-465, 1983. LETHBRIDGE, G.A., BULL, A.T., BURNS, R.G.: Assay and properties of @-1,3-glucanase in soil. Soil Biol. Biochem. 10: 389-392, 1987. MITCHELL, R., ALEXANDER, A.: Lysis of soil fungi by bacteria. Can.J.Microbiol.9: 169-175, 1963. MITCHELL, R.: Addition of fungal cell-wall components to soil for biological disease control. Phytopathology 53: 1068-1073, 1963. RYsAfl, P., MACURA, J.: The assay of 6-galactosidase in soil. Folia Microbiol. 17: 370-374, 1972. SOTOLOVA, I., JANDERA, A.: Activity of 1,3+-D-glucanase in soil. Folia iicrobiol. 30: 521-524, 1985. TAYLOR, C.B.: The nutritional requirements of the predominant flora of the soil. Proc.Soc.Appl.Bact. 14: 101-102, 1951. VAGNEROVA, K., MACURA, J., EATSKA, V.: Rhizosphere microflora of wheat. Folia Microbiol. 5: 298-310, 1960.
-