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Biochemical nature of root exudates in relation to root rot of wheat—III. Carbohydrate shifts in response to foliar treatments
BIOCHEMICAL NATURE OF ROOT EXUDATES IN RELATION TO ROOT ROT OF WHEAT-III. CARBOHYDRATE SHIFTS IN RESPONSE TO FOLIAR TREATMENTS B. L. JALALI Department of Plant Pathology. Haryana Agricultural University. Hissar. 125004 India (Accqtrd
11 .&glrst 1975)
Summary-The alterations brought about in the carbohydrate spectrum of root exudates of wheat in response to six foliar treatments. and their influence on the fungal population of the rhizosphere have been correlated. Pronounced suppression of ribose, maltose and raffinose exudation, which were liberated abundantly from root-rot infected roots. was rccordcd in response to foliar treatments with chloramphenicol and KCI. C‘hloramphenicol and to a lesser extenl 2.4-D also reduced the rhizosphere population. including the test pathogen H[~lminthospo,i~rrn saticum. Previous and present investigations. therefore. indicate that the composition of root exudates could effectively be altered by foliar sprays. in an attempt to control the activity of Helnwthosporium sh~um, causing root-rot of wheat, in soil.
INTRODUCTION
Root exudation and the resultant rhizosphere effect are the two main factors of great significance in root disease initiation. It is becoming increasingly clear that root exudates stimulate plant pathogens (Jalali and Suryanarayana. 1971. 1972) and many other microorganisms (Vrany et al.. 1962; Schroth and Hildebrand, 1964; Rovira, 1969) in the zone of root influence. However, the problem of controlling the pathogenic microflora ‘in the rhizosphere represents one of the important aspects of biological control of plant root diseases. A major factor affecting the rhizosphere microflora is the plant itself and thus the changes in conditions of plant growth are reflected in the microflora of the rhizosphere. Demonstrations that foliar spraying with various chemicals modifies the spectrum of root exudates and microbiological balance in the rhizosphere zone indicate that this may well be used to inhibit root pathogens or to stimulate a microflora which is beneficial for the growth of the host plant. Jalali and Suryanarayana (1970) reported qualitative and quantitative changes in the amino acids exuded from wheat roots as influenced by different foliar applications. This paper describes further studies of shifts in the carbohydrate spectrum of wheat root exudates in response to six foliar treatments. in an attempt to suppress the activity of Heblrirlthosyo~ilrrlt sariawn Pamm. King and Bakke. the causal agent of root rot disease of wheat. in the zone of root influence.
Aseptic collection
Two Mexican varieties of wheat. S-227 and S-308, were used. Rovira’s (1956) technique for collecting root exudates was slightly modified. Surface-sterilized seeds were transferred aseptically to Petri plates containing water agar and subsequently incubated for 5 days at 25 k 2°C in order to detect contaminated seeds and remove seed exudates. One uncontaminated seedling was transferred to each plant culture unit aseptically. The $ants were subjected to foliar treatments after 7 days growth in the culture units. A volume of 25ml of each foliar solution was made in sterilized distilled Hz0 and sprayed on five replications of each treatment by means of a previously sterilized atomizer using glycerine (1% v/v) as a wetting and spreading agent. The surface of the culture tubes was protected from run-off spray by a polythene cover. On the 21st and 28th day of plant growth in the plant unit the foliar treatments were repeated. The controls were sprayed with sterile distilled HZ0 and glycerine. Forty-eight hours after the last spray. the root exudate samples were aseptically collected (Jalali and Suryanarayana. 1970). The root exudate samples, after centrifugation for 30 min at 4OW rev/min- ’ and decanting off the supematant, were concentrated under vacuum to yield 1 ml extract for every 50 plants. Each treatment was replicated three times.
Carhohydratr MATERIALS
Foliar
AND METHODS
sprays
Six chemicals representing fertilizers. antibiotics and plant growth substances were used as foliar sprays. They were: CO(NH,),. (NH&SO,. Na,HPO,, KCl. chloramphenicol and 2.4-dichlorophenoxyacetic acid (2.4-D).
of root exudates
analysis
Because of their high salt content. the exudate samples were desalted by passage through columns of cation-exchange resin Amberlite IR-12O(H+) followed by anion-exchange resin Amberlite IRA-410 (acetate form). The neutral fraction containing the sugars was evaporated to dryness and redissolved in 5 ml 59o isopropanol. The samples were then analysed for carbohydrates (Jalali and Suryanarayana. 1971).
128
B. L.
JAL~LI
Table 1. Effects of foliar sprays on the spectrum of carbohydrate exuded from roots of \\hrat grwn 111ascptlc culture (Results expressed as percentage reduction or increase compared to rhc control \alws~ Foliar Treatments
Carbohydrates
Pentose monosaccharides: Ribose
(NH&SO, (35OmM)
+ 4.3
Xylose
- 15.2
Arabinose
-100
Hexose monosaccharides : Glucose
Chlornmphenicol
Na2HP0, (325 mM)
KC1
CO(NHJ)2
2.4-D
(255mM)
(455mM\
(2.26m.V)
+ 1.8 +9.1 +3,1
- 306 - 8‘1 -9.2
- 13.8 -11-7 -2.3
- 7.2 - 4.8 -8.1
-22-i -9.x - 10.1
Control
(341 m.U) (if8 doplants)
96-w
117~2-1 100~10
-4.5
f3.0
- 23.5
- Il.5
-5.6
Fructose Galactose Rhamnose
+ 3-5 -4.1 + 4.0
- 1.3 -9.0 -100
-9*o +8.0 -100
-5.8 -1-9 -100
+ 3.4 -1.7 - 10.3
-2.1 -2.2 + 12.7 -100
Disaccharides: Maltose Sucrose
- 17.3 -100
- I.7 + 3.27
-100 -100
-100 -100
- 13.7 -ID0
-27.1 -100
92.73 83.7 f
Trisaccharides: Raffinose
-8.4
-6.7
-100
-100
-100
-100
X7.03
Rhizosphere studies
Wheat seeds were sown in small plots (2 x I m) in an experimental area where root rot was of common occurrence. At the 14-day stage of plant growth from seedling emergence. the first foliar applications were given. Control plants wt‘rc sprayed with distilled H,O. Each treatment was replicated three times. Second and third sprays were given at 21-28 days after sowing. To assay the fungal population of the rhizosphere, the soil dilution technique was followed using Czapek’s agar medium. The number of fungal colonies g- ’ oven-dry soil was calculated and the more common fungi were isolated and identified. RESLXTS
Data on the effect of six foliar treatments on the exudation pattern of carbohydrates from wheat roots show (Table 1) that except for the Na,HPO, treatment, exudation of pentose sugars was reduced under all foliar tieatments. KC1 application caused pronounced reduction of glucose and fructose exudation. Complete suppression of rhamnose under ail treatments except (NH&SO, and 2,4-D was of interest. Among disaccharides, release of maltose was not detected under KC1 and CO(NH,), treatments, whereas sucrose exudation was completely suppressed under all foliar treatments except Na*HPO,. Raffinose exudation was not detected under chioramphenicol, 2,4-D, CO(NH,), and KC1 tr~tments. Jalali and Suryanarayana ( 1971) reported pronounced release of ribose. maltose. rafinose and sucrose from wheat roots under the stress of root rot infection. It may, therefore, be seen from the present studies that exudation of three of these four sugars (ribose, maltose and raffinose) could be controlled most effectively by chloramphenicol. KC1 and CO(NH& applications.
147.77 111.33 99.93
i0@0?
The effects of these foliar treatments on the funga1 population of rhizosphere soils of wheat clearly indicated that the fungal population was greatly altered. and such changes varied not only with the chemical sprayed but also with the age of the plant when sprayed (Table 2). In general. most of the treatments reduced the fungal population; however, the first spray with Na2HP04 on 14-day old plants enhanced the population. Among the six foliar treatments given. chloramphenicol application greatly reduced the population. closely followed by 2,4-D treatment. By isolating the commonly occurring fungal genera. it was observed that H. satiour~l could be isolated only under the CO(NH,), and control treatments; under all other treatments the growth of the pathogen seemed to be checked.
DISC1 SSION
lt was suggested by Garrett (19%) that the inlluence exerted by the rhizosphere microftora on the advance of ~~?~~jo~~~~.s ~~~~~~~7j17j.s along wheat roots depends upon the availability of nutrients. Buxton (1957) observed that part of the resistance of peas to attack by soil-borne Fusarim os_r.spor~~r~~may act outside the roots by the differential action of root exudates on the Frrsarizen in the rhizosphere. Higher release of glucose. galactose and oiigosacchdrides from root exudates was observed by Vrany t’t trl. (1961) in response to foliar treatment with chloramphenicol. Jalali and Suryanarayana (1970) showed that exudation of amino acids. which wt’rt congenial for the growth and development of H. sutictw. could bc restricted by various foliar treatments. Foliar sprays thus appear to affect plant metabolism and hence nutrient exudation by roots. Direct trunslo~~~tion of these chemicals to the roots. and their appcarancr in root
Root exudates in relation to root rot pathogcnesis
129
Table 2. ElTects of foliar sprays on the fungal population of rhizosphere and nonrhizosphere
soils of wheat
Fungal population (no. x IO3 g-’ soil)
R
S
Predominant fungal genera
37.01
59.97
4212
a.b.c.d$
69.18
52.1 I
5766
49.89
c.d.e.g
37.12
47.33
39.97
42.69
3509
a.c.d.e
49.23
23.14
54.87
31.16
57.17
29.77
a.b.c.d. e.gh
2.4-D (2.26 mM)
27.07
22.99
35.59
24.94
37.01
24.88
c.e,g
Chloramphenicol (3.41 nlM)
23.97
18.81
20.13
18.17
20.0 I
17.77
c.d.e
Control
63.75
4918
69.95
50.99
78.17
61.65
b.c.d.e. f.g.h
Foliar treatment
First spray (14 days)
Second spray (21 ddyS) S R
Rf
St
4504
21.77
48.99
NasHPOd (325 mM)
66.77
50.38
KC1 (255 mM)
52.99
CO(NHz)l (455 mM)
WK,),SO,
Third spray (28 days)
(350 mM)
l Rhitosphere soil; + Nonrhizosphere soil $a = Alternaria sp.; b = Fusarium sp.; c = Aspergillus sp.; d = Penicillium sp.; e = Mucor sp.; f = Absidia sp.; g = Rhiropus SP.; h = Helminthosporium sativum.
exudates, is also possible. Both effects may have contributed to the results shown in Table 1. It, therefore, seems possible that suitable foliar sprays could lead to the preferential suppression of root pathogens in the zone of root influence. However. it must be stressed that root exudates favouring the pathogen’s survival in soil may not always be correlated with increased pathogenesis since successful parasitism depends on completion of a sequence of steps. One of the many problems in evaluating the response of root exudates in pathogenesis is the diEiculty of setting up experimental models under field conditions with the resultant establishment of natural relationships. Acknowledgements-I
am indebted to Dr. D. Suryanarayana for help and guidance, and to Professor S. D. Garrett, F.R.S. for reading this manuscript.
REFERENCES BUXTONE. W. (1957) Some effects of pea root exudates on physiologic races of Ftrsariuni oxysporm Fr. f. pisi
(Linf.) Snvder & Hansen. Trans. Br. mvcol. Sot. 40. i45-i54. _ GARRET S. D. (1956) Biology of Ifoot Infecting Fungi. Cambridge Universitv Press. Cambridge. JA~LI B. c and SURY~NARAYANA D. (570) Biochemical nature of root exudates in relation to root rot of wheat I. Amino acid shifts in response to foliar treatments. Z. FflKrank.
qjUchrrt_-. 7l
438-442.
JALALIB. L. and SURYANARAYANA D. (1971) Shift in the carbohydrate spectrum of root exudates of wheat in relation to its root rot disease. PI. Soil 34. 261-267. JALALIB. L. and SURYANARAYANA D. (1972) Biochemical nature of root exudates in relation to root rot of wheat II. Amino acid changes. Ind. Phytopath. 25. 195-199. ROVIRAA. D. (1956) Plant root excretions in relation to the rhizosphere effect I. The nature of root exudate from oats and peas. PI. Soil 7. 178-194. ROVIRAA. D. (1969) Plant root exudates. Bot. Rea. 35. 35-57. SCHROTHM. N. and HILDEBRANDD. C. (1964) Influence of plant exudates on root infecting fungi. Ann. Rev. Phytopath. 2. 101-132. VRANYJ.. VANCURAV. V. and MACURAJ. (1962) The effect of foliar applications of some readily metabolized substances, growth regulators and antibiotics on rhizosphere microflora. Folks microbial. 7, 61-70.
11 .&glrst 1975)
Summary-The alterations brought about in the carbohydrate spectrum of root exudates of wheat in response to six foliar treatments. and their influence on the fungal population of the rhizosphere have been correlated. Pronounced suppression of ribose, maltose and raffinose exudation, which were liberated abundantly from root-rot infected roots. was rccordcd in response to foliar treatments with chloramphenicol and KCI. C‘hloramphenicol and to a lesser extenl 2.4-D also reduced the rhizosphere population. including the test pathogen H[~lminthospo,i~rrn saticum. Previous and present investigations. therefore. indicate that the composition of root exudates could effectively be altered by foliar sprays. in an attempt to control the activity of Helnwthosporium sh~um, causing root-rot of wheat, in soil.
INTRODUCTION
Root exudation and the resultant rhizosphere effect are the two main factors of great significance in root disease initiation. It is becoming increasingly clear that root exudates stimulate plant pathogens (Jalali and Suryanarayana. 1971. 1972) and many other microorganisms (Vrany et al.. 1962; Schroth and Hildebrand, 1964; Rovira, 1969) in the zone of root influence. However, the problem of controlling the pathogenic microflora ‘in the rhizosphere represents one of the important aspects of biological control of plant root diseases. A major factor affecting the rhizosphere microflora is the plant itself and thus the changes in conditions of plant growth are reflected in the microflora of the rhizosphere. Demonstrations that foliar spraying with various chemicals modifies the spectrum of root exudates and microbiological balance in the rhizosphere zone indicate that this may well be used to inhibit root pathogens or to stimulate a microflora which is beneficial for the growth of the host plant. Jalali and Suryanarayana (1970) reported qualitative and quantitative changes in the amino acids exuded from wheat roots as influenced by different foliar applications. This paper describes further studies of shifts in the carbohydrate spectrum of wheat root exudates in response to six foliar treatments. in an attempt to suppress the activity of Heblrirlthosyo~ilrrlt sariawn Pamm. King and Bakke. the causal agent of root rot disease of wheat. in the zone of root influence.
Aseptic collection
Two Mexican varieties of wheat. S-227 and S-308, were used. Rovira’s (1956) technique for collecting root exudates was slightly modified. Surface-sterilized seeds were transferred aseptically to Petri plates containing water agar and subsequently incubated for 5 days at 25 k 2°C in order to detect contaminated seeds and remove seed exudates. One uncontaminated seedling was transferred to each plant culture unit aseptically. The $ants were subjected to foliar treatments after 7 days growth in the culture units. A volume of 25ml of each foliar solution was made in sterilized distilled Hz0 and sprayed on five replications of each treatment by means of a previously sterilized atomizer using glycerine (1% v/v) as a wetting and spreading agent. The surface of the culture tubes was protected from run-off spray by a polythene cover. On the 21st and 28th day of plant growth in the plant unit the foliar treatments were repeated. The controls were sprayed with sterile distilled HZ0 and glycerine. Forty-eight hours after the last spray. the root exudate samples were aseptically collected (Jalali and Suryanarayana. 1970). The root exudate samples, after centrifugation for 30 min at 4OW rev/min- ’ and decanting off the supematant, were concentrated under vacuum to yield 1 ml extract for every 50 plants. Each treatment was replicated three times.
Carhohydratr MATERIALS
Foliar
AND METHODS
sprays
Six chemicals representing fertilizers. antibiotics and plant growth substances were used as foliar sprays. They were: CO(NH,),. (NH&SO,. Na,HPO,, KCl. chloramphenicol and 2.4-dichlorophenoxyacetic acid (2.4-D).
of root exudates
analysis
Because of their high salt content. the exudate samples were desalted by passage through columns of cation-exchange resin Amberlite IR-12O(H+) followed by anion-exchange resin Amberlite IRA-410 (acetate form). The neutral fraction containing the sugars was evaporated to dryness and redissolved in 5 ml 59o isopropanol. The samples were then analysed for carbohydrates (Jalali and Suryanarayana. 1971).
128
B. L.
JAL~LI
Table 1. Effects of foliar sprays on the spectrum of carbohydrate exuded from roots of \\hrat grwn 111ascptlc culture (Results expressed as percentage reduction or increase compared to rhc control \alws~ Foliar Treatments
Carbohydrates
Pentose monosaccharides: Ribose
(NH&SO, (35OmM)
+ 4.3
Xylose
- 15.2
Arabinose
-100
Hexose monosaccharides : Glucose
Chlornmphenicol
Na2HP0, (325 mM)
KC1
CO(NHJ)2
2.4-D
(255mM)
(455mM\
(2.26m.V)
+ 1.8 +9.1 +3,1
- 306 - 8‘1 -9.2
- 13.8 -11-7 -2.3
- 7.2 - 4.8 -8.1
-22-i -9.x - 10.1
Control
(341 m.U) (if8 doplants)
96-w
117~2-1 100~10
-4.5
f3.0
- 23.5
- Il.5
-5.6
Fructose Galactose Rhamnose
+ 3-5 -4.1 + 4.0
- 1.3 -9.0 -100
-9*o +8.0 -100
-5.8 -1-9 -100
+ 3.4 -1.7 - 10.3
-2.1 -2.2 + 12.7 -100
Disaccharides: Maltose Sucrose
- 17.3 -100
- I.7 + 3.27
-100 -100
-100 -100
- 13.7 -ID0
-27.1 -100
92.73 83.7 f
Trisaccharides: Raffinose
-8.4
-6.7
-100
-100
-100
-100
X7.03
Rhizosphere studies
Wheat seeds were sown in small plots (2 x I m) in an experimental area where root rot was of common occurrence. At the 14-day stage of plant growth from seedling emergence. the first foliar applications were given. Control plants wt‘rc sprayed with distilled H,O. Each treatment was replicated three times. Second and third sprays were given at 21-28 days after sowing. To assay the fungal population of the rhizosphere, the soil dilution technique was followed using Czapek’s agar medium. The number of fungal colonies g- ’ oven-dry soil was calculated and the more common fungi were isolated and identified. RESLXTS
Data on the effect of six foliar treatments on the exudation pattern of carbohydrates from wheat roots show (Table 1) that except for the Na,HPO, treatment, exudation of pentose sugars was reduced under all foliar tieatments. KC1 application caused pronounced reduction of glucose and fructose exudation. Complete suppression of rhamnose under ail treatments except (NH&SO, and 2,4-D was of interest. Among disaccharides, release of maltose was not detected under KC1 and CO(NH,), treatments, whereas sucrose exudation was completely suppressed under all foliar treatments except Na*HPO,. Raffinose exudation was not detected under chioramphenicol, 2,4-D, CO(NH,), and KC1 tr~tments. Jalali and Suryanarayana ( 1971) reported pronounced release of ribose. maltose. rafinose and sucrose from wheat roots under the stress of root rot infection. It may, therefore, be seen from the present studies that exudation of three of these four sugars (ribose, maltose and raffinose) could be controlled most effectively by chloramphenicol. KC1 and CO(NH& applications.
147.77 111.33 99.93
i0@0?
The effects of these foliar treatments on the funga1 population of rhizosphere soils of wheat clearly indicated that the fungal population was greatly altered. and such changes varied not only with the chemical sprayed but also with the age of the plant when sprayed (Table 2). In general. most of the treatments reduced the fungal population; however, the first spray with Na2HP04 on 14-day old plants enhanced the population. Among the six foliar treatments given. chloramphenicol application greatly reduced the population. closely followed by 2,4-D treatment. By isolating the commonly occurring fungal genera. it was observed that H. satiour~l could be isolated only under the CO(NH,), and control treatments; under all other treatments the growth of the pathogen seemed to be checked.
DISC1 SSION
lt was suggested by Garrett (19%) that the inlluence exerted by the rhizosphere microftora on the advance of ~~?~~jo~~~~.s ~~~~~~~7j17j.s along wheat roots depends upon the availability of nutrients. Buxton (1957) observed that part of the resistance of peas to attack by soil-borne Fusarim os_r.spor~~r~~may act outside the roots by the differential action of root exudates on the Frrsarizen in the rhizosphere. Higher release of glucose. galactose and oiigosacchdrides from root exudates was observed by Vrany t’t trl. (1961) in response to foliar treatment with chloramphenicol. Jalali and Suryanarayana (1970) showed that exudation of amino acids. which wt’rt congenial for the growth and development of H. sutictw. could bc restricted by various foliar treatments. Foliar sprays thus appear to affect plant metabolism and hence nutrient exudation by roots. Direct trunslo~~~tion of these chemicals to the roots. and their appcarancr in root
Root exudates in relation to root rot pathogcnesis
129
Table 2. ElTects of foliar sprays on the fungal population of rhizosphere and nonrhizosphere
soils of wheat
Fungal population (no. x IO3 g-’ soil)
R
S
Predominant fungal genera
37.01
59.97
4212
a.b.c.d$
69.18
52.1 I
5766
49.89
c.d.e.g
37.12
47.33
39.97
42.69
3509
a.c.d.e
49.23
23.14
54.87
31.16
57.17
29.77
a.b.c.d. e.gh
2.4-D (2.26 mM)
27.07
22.99
35.59
24.94
37.01
24.88
c.e,g
Chloramphenicol (3.41 nlM)
23.97
18.81
20.13
18.17
20.0 I
17.77
c.d.e
Control
63.75
4918
69.95
50.99
78.17
61.65
b.c.d.e. f.g.h
Foliar treatment
First spray (14 days)
Second spray (21 ddyS) S R
Rf
St
4504
21.77
48.99
NasHPOd (325 mM)
66.77
50.38
KC1 (255 mM)
52.99
CO(NHz)l (455 mM)
WK,),SO,
Third spray (28 days)
(350 mM)
l Rhitosphere soil; + Nonrhizosphere soil $a = Alternaria sp.; b = Fusarium sp.; c = Aspergillus sp.; d = Penicillium sp.; e = Mucor sp.; f = Absidia sp.; g = Rhiropus SP.; h = Helminthosporium sativum.
exudates, is also possible. Both effects may have contributed to the results shown in Table 1. It, therefore, seems possible that suitable foliar sprays could lead to the preferential suppression of root pathogens in the zone of root influence. However. it must be stressed that root exudates favouring the pathogen’s survival in soil may not always be correlated with increased pathogenesis since successful parasitism depends on completion of a sequence of steps. One of the many problems in evaluating the response of root exudates in pathogenesis is the diEiculty of setting up experimental models under field conditions with the resultant establishment of natural relationships. Acknowledgements-I
am indebted to Dr. D. Suryanarayana for help and guidance, and to Professor S. D. Garrett, F.R.S. for reading this manuscript.
REFERENCES BUXTONE. W. (1957) Some effects of pea root exudates on physiologic races of Ftrsariuni oxysporm Fr. f. pisi
(Linf.) Snvder & Hansen. Trans. Br. mvcol. Sot. 40. i45-i54. _ GARRET S. D. (1956) Biology of Ifoot Infecting Fungi. Cambridge Universitv Press. Cambridge. JA~LI B. c and SURY~NARAYANA D. (570) Biochemical nature of root exudates in relation to root rot of wheat I. Amino acid shifts in response to foliar treatments. Z. FflKrank.
qjUchrrt_-. 7l
438-442.
JALALIB. L. and SURYANARAYANA D. (1971) Shift in the carbohydrate spectrum of root exudates of wheat in relation to its root rot disease. PI. Soil 34. 261-267. JALALIB. L. and SURYANARAYANA D. (1972) Biochemical nature of root exudates in relation to root rot of wheat II. Amino acid changes. Ind. Phytopath. 25. 195-199. ROVIRAA. D. (1956) Plant root excretions in relation to the rhizosphere effect I. The nature of root exudate from oats and peas. PI. Soil 7. 178-194. ROVIRAA. D. (1969) Plant root exudates. Bot. Rea. 35. 35-57. SCHROTHM. N. and HILDEBRANDD. C. (1964) Influence of plant exudates on root infecting fungi. Ann. Rev. Phytopath. 2. 101-132. VRANYJ.. VANCURAV. V. and MACURAJ. (1962) The effect of foliar applications of some readily metabolized substances, growth regulators and antibiotics on rhizosphere microflora. Folks microbial. 7, 61-70.