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Death of rhizobia on inoculated seed
0038-071?/82/010013-02M3.WID
Soil f%>/.B&hem. Vol. 14. pp. 13 10 14. 1982
Copyright
Printed in Great Bntain. Ail rights reserved
DEATH
OF RHIZOBIA M. P.
Department
ON INOCULATED
SALEMA, C. A. PARKER
and D. K,
of Soil Science and Plant Nutrition, University Nedlands, Western Australia 6009
0
1982 Pcrgamon Press Ltd
SEED
KIDBY of Western
Australia,
and
D. L. Western
CHATEL.
Australian Department of Agriculture, Jarrah Road, South Perth, Western Australia 6151 (Accepted
1 J&J- 1981)
Summary-The death of R. trifolii WUI and R. me&xi WU96 was studied with and without protection during the first hour after inoculation on mung bean (Vignu rudiataf seeds. For unprotected rhizobia. death occurred in four distinct phases: a first phase of relatively slow death rate while the seeds remained moist; a second phase of very rapid death shortly after loss of visible moisture on seed; a third phase where numbers tended to stabilize. and a fourth phase in which a significant death rate continued. The use of protectant greatly reduced the overall death rate. especially the rapid death following loss of visible moisture. Some possible mechanisms of cell death and protection are discussed.
INTRODUCTION
R~j~~~i#~, like other Gram-negative bacteria, die rapidly on desiccation. Most of this death has been shown to take place during the initial stages of storage of seeds (Vincent er al., 1962; Burton, 1967). Greenwood (quoted by Vincent, 1959) reported extremely rapid early death in which numbers fell lOOO-fold in 24 h. It was further shown by Burton (196’7)that the highest death occurs in the first hour after inoculation. Our observations with R. trifolii and R. melibri confirm and extend these earlier findings. It was considered important to look at early events following inoculation of seeds in order to better understand the mechanism of cell death. It was of further interest to determine how a protectant against death of rhizobia on seed (Salema et al., 1979) influenced death during the early stages after inoculation. MATERiALS
AND METHODS
Cultures of two strains of fat-lowing rhizobia, R. and R. melifoti WU96 (=U45) were grown in 25 ml Yeast Mannitol Broth (YMB) in 250 ml Erlenmeyer flasks plugged with cotton wool in a rotary incubator at 150rev min-’ and 26’C for 48 h. The broth contained (I-): mannitol, 2g; glucose, 3 g; sucrose, Sg; K2HPOe, 0.5g; NaC1, 0.1 g; MgS0,*7H20, 0.2g; CaS04..2H20, 50mg; NH&l, 0.1 g and Yeast Extract Powder (Difco) 1 g. Twenty grams of agar (Difco certified) were added per litre in the case of solid Yeast Mannitol Agar (YMA). Mung bean (V&a radiura) seeds were used as test surfaces; 0.15 ml of broth culture mixed with 0.35 ml of suspending medium was used to inoculate about 200 surface-sterilized seeds. The suspending media were (i) 29; methyl cellulose or methocel (BDH High substitution), and (ii) a mixture consisting of 10% sucrose and 100; sodium glutamate in 2”/:,methocel. Methocel was used as adhesive and the mixture of sucrose and
sodium glutamate as a protectant of rhizobia against death on seed (Salema et ul., 1979). All the concentrations were on weight of solute to weight of solution basis. Inoculated seeds were left in an open Petri-dish in a laminar flow cabinet and samples were taken at various stages during the drying process in the first hour after inoculation. One sample was taken at each sampling time. Each sample consisted of 10 seeds from which the rhizobia were thoroughly suspended in 10 ml sterile YMB in 30 ml McCartney bottles by hand shaking for 5 min. The Miles and Misra drop count technique (Vincent, 1970) was used for counting after making necessary lO-fold serial dilutions. Three drops from each dilution were plated on each of 3 YMA plates; i.e. a total of 9 drops for every dilution. Colony counts from a suitable dilution were made from each of the 9 drops after incubating the plates at 26-C for 3-4 days. The final count from each sample was calculated from the average number of colonies per drop. RESULTS
rrifolii WUl (=TAl)
The survival curves of R. trifolii and R. meliloti during the first hour after inoculation, with and without protection, are shown in Figs 1 and 2, respectively. The results show that when rhizobia on seed were unprotected, death occurred in distinct phases. There was a phase of relatively slow death while the seeds remained moist, i.e. visible free water, foltowed by a very high death rate phase shortly after loss of visible moisture. Following the second phase, numbers tended to stabilize for about 15 min after which a significant death rate continued. When rhizobia were protected, the overall death rate was reduced, especially rapid death following loss of visible moisture. DISCUSSION It has not been possible to clearly demonstrate whether death of rhizobia on seed is primarily due to
13
POINT OF LOSS OF
2% METHOCEL 10% SUCROSE
/ /
I 10
361 0
’ 2 % METHOCEL \
;I 20
MINUTES
I 30
AFTER
I LO
I 50
I 60
INOCULATION
Fig. 1. Survival on seed of unprotected and protected R. trifi,/ii WUl during the first hour after inoculation (S’,, SE shown).
rated when put into YMB used for washing them off the seeds in order to count them. The rapid death shortly after the point of loss of visible moisture suggests that both desiccation and rehydration are causing death. Alternate slow and rapid death similar to that exhibited by unprotected rhizobia in this study has been shown in Sulmone//u by Moats ef ul. (1971). They attributed this to the heterogenous nature of the bacterial culture population. The main cause of injury to rhizobia due to drying and rehydration is suggested to be leakage of important cell constituents (Bushby and Marshall, 1977). Such leakage is indicative of membrane damage and has been explained for plant cells in terms of changes which take place in the structure of membranes during desiccation (Simon. 1974). Membrane components may become displaced by the rapid influx of water. resulting in leakage of cell components. This could be an explanation of the rapid death following loss of visible moisture. Integrity of desiccated structural proteins and other macromolecular complexes can be retained if sufficient water remains in association with them (Hsiao, 1973). The protectant referred to in this experiment could have improved survival by maintaining a sufficient bound water content in the cell envelope. It is also possible that the protectant was able to functionally replace the water lost during desiccation and thus prevented the formation of unfavourable conformations in labile structures. Ac!ino~let/yrmm~-We are grateful for financial support given to M. P. Salema by the Australian Government under the Special Commonwealth African Assistance Plan.
POINT OF LOSS OF IVISIBLE MOISTURE
REFERENCES
50
I
0
I I
.L I
I
I
I
10 20 30 LO 50 MINUTES AFTER INOCULATION
Fig. 2. Survival on seed of unprotected
1 60
and protected R. (5””
ntelilori WU96 during the first hour after inoculation
SE shown).
the effect of desiccation or the effect of rehydration, or both, because all the methods of determining viability involve rehydration. In this study, for unprotected rhizobia, death was minimal up to the point of loss of visible moisture. At the point of loss of visible moisture the cells were “dry” and were therefore rehyd-
BURTONJ. (1967) Rhixbium culture and use. In Micwbitrl Technology (H. J. Peppler, Ed.), pp. l-33. Van NostrandReinhold. New York. BUSHBYH. V. A. and MARSHALLK. C. (1977) Desiccation induced damage to the cell envelope of root-nodule bacteria. Soil Biology & Biuchemistrr 9, 149- 152. HSIAOT. C. (1973) Plant responses to water stress. Annut// Reriew of Plunt Physiology 24, 519-570. MOATSW. A.. DA~BAH R. and EDWARDSW. M. (1971) Interpretation of nonlo~~rithmie survivor curves of heateh bacteria. Juurn~~ 2 Food Science 36, 523-526. SALEMAM. P.. PARKERC. A. and KIDBYD. K. (1979) The survival of &-_obium cells on seed. In Proceedings of the 6th rlustralian Nodulution Conference, pp. 50-53. SIMONE. W. (1978) Membranes in dry and imbibing seeds. In Dry Bioluyicul Systems (J. H. Crowe and J. S. Clegg, Eds), pp. 205-224. Academic Press, New York. VINCENTJ. M. (1959) Survival of the root-nodule bacteria. in Nutrition of the Legmes (E. G. Hallsworth. Ed.), pp. 108-123. Academic P&s. New York. VINCENTJ. M., THOMPSON J. A. and DONOVANK. 0. (1962) Death of root nodule bacteria on drying. Austrulian Journtd of Agricultural
Resew&
13, 258-270.
VINCENTJ. M. (19701 9 ~~~~~t~~~~~ the Prctcticai Study of Root-Nodli~e Bacteriti. IBP ~and~#~~ No. ij. Blackwe~l, Oxford.
Soil f%>/.B&hem. Vol. 14. pp. 13 10 14. 1982
Copyright
Printed in Great Bntain. Ail rights reserved
DEATH
OF RHIZOBIA M. P.
Department
ON INOCULATED
SALEMA, C. A. PARKER
and D. K,
of Soil Science and Plant Nutrition, University Nedlands, Western Australia 6009
0
1982 Pcrgamon Press Ltd
SEED
KIDBY of Western
Australia,
and
D. L. Western
CHATEL.
Australian Department of Agriculture, Jarrah Road, South Perth, Western Australia 6151 (Accepted
1 J&J- 1981)
Summary-The death of R. trifolii WUI and R. me&xi WU96 was studied with and without protection during the first hour after inoculation on mung bean (Vignu rudiataf seeds. For unprotected rhizobia. death occurred in four distinct phases: a first phase of relatively slow death rate while the seeds remained moist; a second phase of very rapid death shortly after loss of visible moisture on seed; a third phase where numbers tended to stabilize. and a fourth phase in which a significant death rate continued. The use of protectant greatly reduced the overall death rate. especially the rapid death following loss of visible moisture. Some possible mechanisms of cell death and protection are discussed.
INTRODUCTION
R~j~~~i#~, like other Gram-negative bacteria, die rapidly on desiccation. Most of this death has been shown to take place during the initial stages of storage of seeds (Vincent er al., 1962; Burton, 1967). Greenwood (quoted by Vincent, 1959) reported extremely rapid early death in which numbers fell lOOO-fold in 24 h. It was further shown by Burton (196’7)that the highest death occurs in the first hour after inoculation. Our observations with R. trifolii and R. melibri confirm and extend these earlier findings. It was considered important to look at early events following inoculation of seeds in order to better understand the mechanism of cell death. It was of further interest to determine how a protectant against death of rhizobia on seed (Salema et al., 1979) influenced death during the early stages after inoculation. MATERiALS
AND METHODS
Cultures of two strains of fat-lowing rhizobia, R. and R. melifoti WU96 (=U45) were grown in 25 ml Yeast Mannitol Broth (YMB) in 250 ml Erlenmeyer flasks plugged with cotton wool in a rotary incubator at 150rev min-’ and 26’C for 48 h. The broth contained (I-): mannitol, 2g; glucose, 3 g; sucrose, Sg; K2HPOe, 0.5g; NaC1, 0.1 g; MgS0,*7H20, 0.2g; CaS04..2H20, 50mg; NH&l, 0.1 g and Yeast Extract Powder (Difco) 1 g. Twenty grams of agar (Difco certified) were added per litre in the case of solid Yeast Mannitol Agar (YMA). Mung bean (V&a radiura) seeds were used as test surfaces; 0.15 ml of broth culture mixed with 0.35 ml of suspending medium was used to inoculate about 200 surface-sterilized seeds. The suspending media were (i) 29; methyl cellulose or methocel (BDH High substitution), and (ii) a mixture consisting of 10% sucrose and 100; sodium glutamate in 2”/:,methocel. Methocel was used as adhesive and the mixture of sucrose and
sodium glutamate as a protectant of rhizobia against death on seed (Salema et ul., 1979). All the concentrations were on weight of solute to weight of solution basis. Inoculated seeds were left in an open Petri-dish in a laminar flow cabinet and samples were taken at various stages during the drying process in the first hour after inoculation. One sample was taken at each sampling time. Each sample consisted of 10 seeds from which the rhizobia were thoroughly suspended in 10 ml sterile YMB in 30 ml McCartney bottles by hand shaking for 5 min. The Miles and Misra drop count technique (Vincent, 1970) was used for counting after making necessary lO-fold serial dilutions. Three drops from each dilution were plated on each of 3 YMA plates; i.e. a total of 9 drops for every dilution. Colony counts from a suitable dilution were made from each of the 9 drops after incubating the plates at 26-C for 3-4 days. The final count from each sample was calculated from the average number of colonies per drop. RESULTS
rrifolii WUl (=TAl)
The survival curves of R. trifolii and R. meliloti during the first hour after inoculation, with and without protection, are shown in Figs 1 and 2, respectively. The results show that when rhizobia on seed were unprotected, death occurred in distinct phases. There was a phase of relatively slow death while the seeds remained moist, i.e. visible free water, foltowed by a very high death rate phase shortly after loss of visible moisture. Following the second phase, numbers tended to stabilize for about 15 min after which a significant death rate continued. When rhizobia were protected, the overall death rate was reduced, especially rapid death following loss of visible moisture. DISCUSSION It has not been possible to clearly demonstrate whether death of rhizobia on seed is primarily due to
13
POINT OF LOSS OF
2% METHOCEL 10% SUCROSE
/ /
I 10
361 0
’ 2 % METHOCEL \
;I 20
MINUTES
I 30
AFTER
I LO
I 50
I 60
INOCULATION
Fig. 1. Survival on seed of unprotected and protected R. trifi,/ii WUl during the first hour after inoculation (S’,, SE shown).
rated when put into YMB used for washing them off the seeds in order to count them. The rapid death shortly after the point of loss of visible moisture suggests that both desiccation and rehydration are causing death. Alternate slow and rapid death similar to that exhibited by unprotected rhizobia in this study has been shown in Sulmone//u by Moats ef ul. (1971). They attributed this to the heterogenous nature of the bacterial culture population. The main cause of injury to rhizobia due to drying and rehydration is suggested to be leakage of important cell constituents (Bushby and Marshall, 1977). Such leakage is indicative of membrane damage and has been explained for plant cells in terms of changes which take place in the structure of membranes during desiccation (Simon. 1974). Membrane components may become displaced by the rapid influx of water. resulting in leakage of cell components. This could be an explanation of the rapid death following loss of visible moisture. Integrity of desiccated structural proteins and other macromolecular complexes can be retained if sufficient water remains in association with them (Hsiao, 1973). The protectant referred to in this experiment could have improved survival by maintaining a sufficient bound water content in the cell envelope. It is also possible that the protectant was able to functionally replace the water lost during desiccation and thus prevented the formation of unfavourable conformations in labile structures. Ac!ino~let/yrmm~-We are grateful for financial support given to M. P. Salema by the Australian Government under the Special Commonwealth African Assistance Plan.
POINT OF LOSS OF IVISIBLE MOISTURE
REFERENCES
50
I
0
I I
.L I
I
I
I
10 20 30 LO 50 MINUTES AFTER INOCULATION
Fig. 2. Survival on seed of unprotected
1 60
and protected R. (5””
ntelilori WU96 during the first hour after inoculation
SE shown).
the effect of desiccation or the effect of rehydration, or both, because all the methods of determining viability involve rehydration. In this study, for unprotected rhizobia, death was minimal up to the point of loss of visible moisture. At the point of loss of visible moisture the cells were “dry” and were therefore rehyd-
BURTONJ. (1967) Rhixbium culture and use. In Micwbitrl Technology (H. J. Peppler, Ed.), pp. l-33. Van NostrandReinhold. New York. BUSHBYH. V. A. and MARSHALLK. C. (1977) Desiccation induced damage to the cell envelope of root-nodule bacteria. Soil Biology & Biuchemistrr 9, 149- 152. HSIAOT. C. (1973) Plant responses to water stress. Annut// Reriew of Plunt Physiology 24, 519-570. MOATSW. A.. DA~BAH R. and EDWARDSW. M. (1971) Interpretation of nonlo~~rithmie survivor curves of heateh bacteria. Juurn~~ 2 Food Science 36, 523-526. SALEMAM. P.. PARKERC. A. and KIDBYD. K. (1979) The survival of &-_obium cells on seed. In Proceedings of the 6th rlustralian Nodulution Conference, pp. 50-53. SIMONE. W. (1978) Membranes in dry and imbibing seeds. In Dry Bioluyicul Systems (J. H. Crowe and J. S. Clegg, Eds), pp. 205-224. Academic Press, New York. VINCENTJ. M. (1959) Survival of the root-nodule bacteria. in Nutrition of the Legmes (E. G. Hallsworth. Ed.), pp. 108-123. Academic P&s. New York. VINCENTJ. M., THOMPSON J. A. and DONOVANK. 0. (1962) Death of root nodule bacteria on drying. Austrulian Journtd of Agricultural
Resew&
13, 258-270.
VINCENTJ. M. (19701 9 ~~~~~t~~~~~ the Prctcticai Study of Root-Nodli~e Bacteriti. IBP ~and~#~~ No. ij. Blackwe~l, Oxford.