- Email: [email protected]
Survival of Rhizobium japonicum in various carriers
Zbl. Bakt. Abt. II, Bd.128, S. 232-239 (1973)
[Division of Microbiology, Indian Agricultural Research Institute, New Delhi-12, India]
Survival of Rhizobium japonicum in various carriers Maitrayee Pramanik and V.Iswaran With 4 figures
Summary Study on longevity of Rhizobium japonicum maintained at 29°e, in carriers such as peat, farm-yard manure, charcoal, and hay, mixed each with varying proportions of garden soil, was followed over a period of 12 weeks with determination of the rhizobial content once in a fortnight. The utility of the carrier, judged on the basis of a minimum count of 106 rhizobia per gram at the end of 12 weeks of incubation, indicated 1. Higher concentration of peat and charcoal were superior to other carriers.
2. The decline of rhizobiaI population in carriers where concentration of peat or charcoal was 50% and above, was not as rapid as in the control soil. 3. Soil amended with FYM was better at a concentration lower than 50%. To the production of legume inoculants on a commercial scale, several workers in the past reported finely ground peat as a satisfactory carrier for Rhizobium (LOCHHEAD and THEXTON 1947, REDLIN and NEWTON 1948, NEWBOULD 1951, DATE 1959, BURTON 1960, SANKARAM 1960, VINCENT 1962 and 1965, BROCKWELL and PHILLIPS 1965, ISWARAN et al. 1969). Recently, materials like lignite (KANDASAMY and PRASAD 1971) and cellulose powder (PUGASHETTI et al. 1971) have also been found to be good carriers. Charcoal as an alternative choice found favour first due to its capacity for absorption of toxic compounds, secreted by the roots or seed coat ofthe legume, accompanied by a favourable reaction and aeration of the medium conducive for enhancing the longevity of the bacteria (ZINDZADZE 1932, PAPADAKIS 1941, TURNER 1955, RELY et al. 1957, PATTRICK and KOCH 1958). Farmyard manure, for its organic matter content, was regarded as another alternate (GUKOVA and BUTKEVICH 1941, TINCKER 1947). Although peat is regarded as most satisfactory, its absence both in quantity and quality in India necessiated the search for alternative sources that would primarily allow the organism to remain viable over a reasonable period, and the results of study to this end are reported below.
Survival of Rhizobium japonicum in various carriers
233
Materials and Methods
Inoculant strains: The strain of Rhizobium japonicum, used for inoculating the various carriers, was that which is maintained in the Type-culture collection of the Division of Microbiology, Indian Agricultural Research Institute, New Delhi. Carriers: The following carriers were used: (1) Peat (Nilgiri Hills), (2) FYM or compost, (3) charcoal (wood), and (4) hay (rice). Each carrier was mixed with garden soil to obtain a final concentration of 10, 25, 50, and 100 per cent. As higher concentrations of hay were found to be toxic to the test organism, 1 and 5 per cent were also used in addition. Soil alone was used as a control. Drying and sieving: The samples were all dried at 60-80 DC for 24 hours. The hay was sieved through a 0.1 mm sieve, while in the case of soil, FYM, and peat a 0.25 mm sieve was used. Liquid culture: The soybean culture was prepared by inoculating in the usual Yeast-extractmannitol medium (FRED et al. 1932) and shaking in a rotary shaker for 7 days at a temperature of 28 °C-30 DC. One hundred grams of the carriers were placed in Erlenmeyer flasks, stoppered with non-absorbent cotton, and sterilized at 121 DC for three hours. Moisture content of the carriers were brought to 40 per cent of the water holding capacity, inclusive of the water carried by the inoculum. After inoculation the flasks, were covered with cellophane paper to prevent contamination and desiccation during their incubation at 29 DC. Bacterial counts: The samples were arranged for bacterial counts by the plate method every fortnight in the YEMA medium, containing Congo-red (1:400) (FRED et al. 1932).
Results and Discussion
Survival values of Rhizobiumjaponicum in soil peat mixture (Fig. 1) show that addition of 10 per cent peat has been found to be more efficient than soil alone. Survival of Rhizobium japonicum in soil FYM mixture (Fig. 2) indicates that the highest count of rhizobia could be observed in 10 per cent FYM at the end ofthe 4th week of storage. By the addition of 50 per cent and more, the rhizobial count decreased very rapidly. As addition in 10 g. in soil showed better result than the control soil, it can be presumed to be due to some essential substances, contained therein (MASEFIELD 1965). The results of Fig. 3 show the survival of Rhizobium japonicum in a soil charcoal mixture. The data show that for a long term storage at room temperature (29°C), addition of charcoal beyond 50 per cent is beneficial for the survival of rhizobia. The death rate is not as rapid as in the control soil. The reason for this may be due to the high adsorptive capacity of both amendments and as well as toxic substances by the charcoal as well. Repeated addition of charcoal facilitates the bacterial metabolism (GORELICK et al. 1951). The results are in agreement with the findings of NEWBOULD (1951) who obtained the highest number of R. meliloti in a mixture of 50: 50 of peat and powdered charcoal to which nutrient solution was added. Survival of R. japonicum in hay (Fig. 4) show that higher concentration of hay is toxic to rhizobia. In the present study it was observed that addition of hay beyond the 5 per cent level was toxic to this rhizobia, while R. trifolii have been shown to tolerate at least 10 per cent concentration. This shows that R.japonicum is more sensitive than R. trifolii. This has been further examplified by the correlation coefficient which has been found to be the highest, thus indicating that the toxicity increases with the increase in concentration of hay. ISWARAN and HARRIS (1968) showed that this toxicity may be due to the liberation of polyphenols and organic acids during the decomposition of hay. The toxicity was greater in legumes than in cereals.
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Fig,2, Survival of Rhizobium japonicum
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Fig.3. Survival of Rhizobium japonicum
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Fig.4. Survival of Rhizobium japonicum
Conclusions
This study on the longevity of R. japonicum in sterilized carriers such as peat, farmyard manure (FYM), charcoal, and hay has shown that (1) higher concentrations of peat and charcoal were definitely superior to other test materials. (2) The decay rate of the blwterial cells in carriers, where the concentration of peat and charcoal were 50 per cent or above, was not as rapid as in the case of soil alone. (3) The correlation coefficient of the
238
:Maitrayee Pramanik and V. Iswaran
survival of R. japonicum in hay was found to be the highest, indicating the toxic effect of hay with increase in concentration. The authors thank Dr. W. V.B. Sundara, Rao, former Head of the Division of :Microbiology, his interest and encouragement.
Zusammenfassung Del' EinfluB von unterschiedlichen Zusiitzen (Torf, Stallmist, Holzkohle und Heu) in unterschiedlichen :Mengen zu Gartenerde wurde untersucht. 'Viihrend 12 Wochen wurde jede zweite Woche die Rhizobienzahl ermittelt. Es hat sich folgendes ergeben: 1. Eine hiihere Konzentration von Torf und Holzkohle war den anderen Zusiitzen iiberlegen. 2. Die Abnahme del' Rhizobienzahl war in den Priiparaten mit 50% Torf odeI' Holzkohle geringer als in Kontrollerde. 3. Beim Stalldungzusatz liegt die optimale Zusatzmenge unter 50%. Literatur BROCKWELL, J., and PHILLIPS, L. J.: Survival at high temperature of Rhizobium meliloti in peat inoculant on lucerne seed. Aust. J. Sci. 27 (1965), 332. - BURTON, J. C.: Pre-inoculated seed-good or bad. (Personal communication). (1960). - DATE, RA.: Survival oflucerne rhizobia in laboratory peat. (Personal communication). (1959). - DILZ, K., and :MULDER, E. G.: The effect of soil pH stable manure and fertilizer nitrogen on the growth of red-clover and of red-clover associations with perennial rye grass. Netherlands J. Agri. Sci. 10 (1962), 1. - FRED, E. B., BALDWIN, I. L., and :McCOY, K: Root nodule Bacteria and Leguminous plants. Univ. of. Wisconsin. Studies in Sci. Nos. :Madison, Wisconsin (1932). - GORELICK. A.N., :MEAD, D.D., and KELLY, KH.: The growth of bacteria in a charcoal cellophane system. J. Bact. 61 (1951), 507.GREENWOOD, R.:M.: Effect of cultivation and animal oung on rhizobial numbers (Personal communication). (1958). - GUKOVA, :M.:M., and BUTKEVITCH, W. S.: Influence of aeration and soil temperature on the development of inoculated and non-inoculated soya plant. C.R. Acad. Sci. U. S. S.R (N. S.) 31 (1941), 437. - HEDLIN, RA., and NEWTON, J.D.: Some factors influencing growth and survival of Rhizobium in humus and soil culture. Can. J. Res. 26c(1948), 174. - RELY, F. W., BERGERSEN, I. J., and BI(OCKWELL, J.: :Microbial antagonism in the rhizosphere as a factor in the failure of inoculation of the subterranean clover. Aust. J. Agri. Res. 8 (1957), 24. ISWARAN, V., and HARRIS, J.R.: The bacteria of decomposing cereal straw and their effects on plant growth. Ninth Inter. Congo Soil Sci. Trans. 3 (52) (1968), 501. - ISWARAN, V., SUNDARA RAo, W. V. B., :MAGU, S. P., and JAUHRI, K. S.: Indian peat as a carrier of Rhizobium. Curl', Sci. 38 (1969), 468. - KANDASAMY, R, and PRASAD,N.N.: Lignite as a carrier of Rhizobia. Curl'. Sci. 40 (18) (1971), 496. - LOCHHEAD,A.G., and THEXTON,RH.: Growth and survival of bacteria in peat. Can. J. Res. 250c (1947), 1. - :MASEFIELD, G.B.: The effect of organic matter in soil on legume nodulation. Expt. Agri. 1 (2) (1965), 113. - :MULDER, E. G., and VAN VEEN, W. L.: Effect of pH and organic compounds on nitrogen fixation by red-clover. Plant and Soil 13 (2) (1960), 91. - N EWBOULD, F. H. S.: Studies on humus type legume-inoculants. Growth and survival in storage. Sci. Agri. 31 (1951), 463. - PAPADAKIS, J. S.: An important effect of soil colloids on plant growth. Soil Sci. 52 (1941), 283. - PATRICK, Z. A., and KOCH, L. W.: Inhibition of respiration, germination, and growth by substances arising during the decomposition of certain plant residues in the soil. Can. J. Bot. 36 (1958), 621. - PUGASHETTI, B.K., GOPALGOWDA, H. S., and PATH" R B.: Cellulose powder as legume inoculant base. Curro Sci. 40 (18) (1971),494. - SANKARAM, A.: Nature and quality of legume inoculant. Sci. and Culture. 25 (10) (1960), 569. - SINGH, R. S., and SITARAMAIAH, K.: Effect of decomposing green leaves, sawdust, and urea on the incidence of root-knot of okra and tomato. Indian Phytopathol. 20 (4) (1967), 349. - TINCKER, :M. A. H.: Experiments with Lilies at Wisley. VI. A summary of observation made concerning Lily roots and soil conditions. The Lily-year-book. Roy. Hort. Soc.
Survival of Rhizobium japonicum in various carriers
239
London. 1947. - TURNER, E. R.: The effect of certain adsorbants on the nodulation of clover plants Ann. Bot. 19 (1955),149. - VINCENT, J.M.: Australian studies of the root nodule bacteria. Proc. Linn. Soc. N.S.W .87 (1962), 8.-VINCENT, J.M.: In "Soil-Nitrogen"; Bartholomew, W. W. and Clark, F.E. (Ed.). Amer. Soc. Agron. Monograph ch. 10 (1965),384. -ZINDZADZE, C.H.: Nutrition artificielle des plantes cultivatus Me'langes nutritifs a pH stable. Ann. Agron. Paris (N. S.) 2 (1932), 809.
Authors' address: Dr. Maitryee Pramanick and Dr. V.Iswaran, Division of Microbiology, Indian Agricultural Research Institute, New Delhi - 12 (INDIA)-
[Division of Microbiology, Indian Agricultural Research Institute, New Delhi-12, India]
Survival of Rhizobium japonicum in various carriers Maitrayee Pramanik and V.Iswaran With 4 figures
Summary Study on longevity of Rhizobium japonicum maintained at 29°e, in carriers such as peat, farm-yard manure, charcoal, and hay, mixed each with varying proportions of garden soil, was followed over a period of 12 weeks with determination of the rhizobial content once in a fortnight. The utility of the carrier, judged on the basis of a minimum count of 106 rhizobia per gram at the end of 12 weeks of incubation, indicated 1. Higher concentration of peat and charcoal were superior to other carriers.
2. The decline of rhizobiaI population in carriers where concentration of peat or charcoal was 50% and above, was not as rapid as in the control soil. 3. Soil amended with FYM was better at a concentration lower than 50%. To the production of legume inoculants on a commercial scale, several workers in the past reported finely ground peat as a satisfactory carrier for Rhizobium (LOCHHEAD and THEXTON 1947, REDLIN and NEWTON 1948, NEWBOULD 1951, DATE 1959, BURTON 1960, SANKARAM 1960, VINCENT 1962 and 1965, BROCKWELL and PHILLIPS 1965, ISWARAN et al. 1969). Recently, materials like lignite (KANDASAMY and PRASAD 1971) and cellulose powder (PUGASHETTI et al. 1971) have also been found to be good carriers. Charcoal as an alternative choice found favour first due to its capacity for absorption of toxic compounds, secreted by the roots or seed coat ofthe legume, accompanied by a favourable reaction and aeration of the medium conducive for enhancing the longevity of the bacteria (ZINDZADZE 1932, PAPADAKIS 1941, TURNER 1955, RELY et al. 1957, PATTRICK and KOCH 1958). Farmyard manure, for its organic matter content, was regarded as another alternate (GUKOVA and BUTKEVICH 1941, TINCKER 1947). Although peat is regarded as most satisfactory, its absence both in quantity and quality in India necessiated the search for alternative sources that would primarily allow the organism to remain viable over a reasonable period, and the results of study to this end are reported below.
Survival of Rhizobium japonicum in various carriers
233
Materials and Methods
Inoculant strains: The strain of Rhizobium japonicum, used for inoculating the various carriers, was that which is maintained in the Type-culture collection of the Division of Microbiology, Indian Agricultural Research Institute, New Delhi. Carriers: The following carriers were used: (1) Peat (Nilgiri Hills), (2) FYM or compost, (3) charcoal (wood), and (4) hay (rice). Each carrier was mixed with garden soil to obtain a final concentration of 10, 25, 50, and 100 per cent. As higher concentrations of hay were found to be toxic to the test organism, 1 and 5 per cent were also used in addition. Soil alone was used as a control. Drying and sieving: The samples were all dried at 60-80 DC for 24 hours. The hay was sieved through a 0.1 mm sieve, while in the case of soil, FYM, and peat a 0.25 mm sieve was used. Liquid culture: The soybean culture was prepared by inoculating in the usual Yeast-extractmannitol medium (FRED et al. 1932) and shaking in a rotary shaker for 7 days at a temperature of 28 °C-30 DC. One hundred grams of the carriers were placed in Erlenmeyer flasks, stoppered with non-absorbent cotton, and sterilized at 121 DC for three hours. Moisture content of the carriers were brought to 40 per cent of the water holding capacity, inclusive of the water carried by the inoculum. After inoculation the flasks, were covered with cellophane paper to prevent contamination and desiccation during their incubation at 29 DC. Bacterial counts: The samples were arranged for bacterial counts by the plate method every fortnight in the YEMA medium, containing Congo-red (1:400) (FRED et al. 1932).
Results and Discussion
Survival values of Rhizobiumjaponicum in soil peat mixture (Fig. 1) show that addition of 10 per cent peat has been found to be more efficient than soil alone. Survival of Rhizobium japonicum in soil FYM mixture (Fig. 2) indicates that the highest count of rhizobia could be observed in 10 per cent FYM at the end ofthe 4th week of storage. By the addition of 50 per cent and more, the rhizobial count decreased very rapidly. As addition in 10 g. in soil showed better result than the control soil, it can be presumed to be due to some essential substances, contained therein (MASEFIELD 1965). The results of Fig. 3 show the survival of Rhizobium japonicum in a soil charcoal mixture. The data show that for a long term storage at room temperature (29°C), addition of charcoal beyond 50 per cent is beneficial for the survival of rhizobia. The death rate is not as rapid as in the control soil. The reason for this may be due to the high adsorptive capacity of both amendments and as well as toxic substances by the charcoal as well. Repeated addition of charcoal facilitates the bacterial metabolism (GORELICK et al. 1951). The results are in agreement with the findings of NEWBOULD (1951) who obtained the highest number of R. meliloti in a mixture of 50: 50 of peat and powdered charcoal to which nutrient solution was added. Survival of R. japonicum in hay (Fig. 4) show that higher concentration of hay is toxic to rhizobia. In the present study it was observed that addition of hay beyond the 5 per cent level was toxic to this rhizobia, while R. trifolii have been shown to tolerate at least 10 per cent concentration. This shows that R.japonicum is more sensitive than R. trifolii. This has been further examplified by the correlation coefficient which has been found to be the highest, thus indicating that the toxicity increases with the increase in concentration of hay. ISWARAN and HARRIS (1968) showed that this toxicity may be due to the liberation of polyphenols and organic acids during the decomposition of hay. The toxicity was greater in legumes than in cereals.
234
Maitrayee Pramanik and V. Iswaran
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Fig.I. Survival of Rhizobium japonicum
IN
WEEKS
I
12
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,
14
235
Survival of Rhizobium japonicum in various carriers
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Fig,2, Survival of Rhizobium japonicum
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/2
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Fig.3. Survival of Rhizobium japonicum
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%
COAL I
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237
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Fig.4. Survival of Rhizobium japonicum
Conclusions
This study on the longevity of R. japonicum in sterilized carriers such as peat, farmyard manure (FYM), charcoal, and hay has shown that (1) higher concentrations of peat and charcoal were definitely superior to other test materials. (2) The decay rate of the blwterial cells in carriers, where the concentration of peat and charcoal were 50 per cent or above, was not as rapid as in the case of soil alone. (3) The correlation coefficient of the
238
:Maitrayee Pramanik and V. Iswaran
survival of R. japonicum in hay was found to be the highest, indicating the toxic effect of hay with increase in concentration. The authors thank Dr. W. V.B. Sundara, Rao, former Head of the Division of :Microbiology, his interest and encouragement.
Zusammenfassung Del' EinfluB von unterschiedlichen Zusiitzen (Torf, Stallmist, Holzkohle und Heu) in unterschiedlichen :Mengen zu Gartenerde wurde untersucht. 'Viihrend 12 Wochen wurde jede zweite Woche die Rhizobienzahl ermittelt. Es hat sich folgendes ergeben: 1. Eine hiihere Konzentration von Torf und Holzkohle war den anderen Zusiitzen iiberlegen. 2. Die Abnahme del' Rhizobienzahl war in den Priiparaten mit 50% Torf odeI' Holzkohle geringer als in Kontrollerde. 3. Beim Stalldungzusatz liegt die optimale Zusatzmenge unter 50%. Literatur BROCKWELL, J., and PHILLIPS, L. J.: Survival at high temperature of Rhizobium meliloti in peat inoculant on lucerne seed. Aust. J. Sci. 27 (1965), 332. - BURTON, J. C.: Pre-inoculated seed-good or bad. (Personal communication). (1960). - DATE, RA.: Survival oflucerne rhizobia in laboratory peat. (Personal communication). (1959). - DILZ, K., and :MULDER, E. G.: The effect of soil pH stable manure and fertilizer nitrogen on the growth of red-clover and of red-clover associations with perennial rye grass. Netherlands J. Agri. Sci. 10 (1962), 1. - FRED, E. B., BALDWIN, I. L., and :McCOY, K: Root nodule Bacteria and Leguminous plants. Univ. of. Wisconsin. Studies in Sci. Nos. :Madison, Wisconsin (1932). - GORELICK. A.N., :MEAD, D.D., and KELLY, KH.: The growth of bacteria in a charcoal cellophane system. J. Bact. 61 (1951), 507.GREENWOOD, R.:M.: Effect of cultivation and animal oung on rhizobial numbers (Personal communication). (1958). - GUKOVA, :M.:M., and BUTKEVITCH, W. S.: Influence of aeration and soil temperature on the development of inoculated and non-inoculated soya plant. C.R. Acad. Sci. U. S. S.R (N. S.) 31 (1941), 437. - HEDLIN, RA., and NEWTON, J.D.: Some factors influencing growth and survival of Rhizobium in humus and soil culture. Can. J. Res. 26c(1948), 174. - RELY, F. W., BERGERSEN, I. J., and BI(OCKWELL, J.: :Microbial antagonism in the rhizosphere as a factor in the failure of inoculation of the subterranean clover. Aust. J. Agri. Res. 8 (1957), 24. ISWARAN, V., and HARRIS, J.R.: The bacteria of decomposing cereal straw and their effects on plant growth. Ninth Inter. Congo Soil Sci. Trans. 3 (52) (1968), 501. - ISWARAN, V., SUNDARA RAo, W. V. B., :MAGU, S. P., and JAUHRI, K. S.: Indian peat as a carrier of Rhizobium. Curl', Sci. 38 (1969), 468. - KANDASAMY, R, and PRASAD,N.N.: Lignite as a carrier of Rhizobia. Curl'. Sci. 40 (18) (1971), 496. - LOCHHEAD,A.G., and THEXTON,RH.: Growth and survival of bacteria in peat. Can. J. Res. 250c (1947), 1. - :MASEFIELD, G.B.: The effect of organic matter in soil on legume nodulation. Expt. Agri. 1 (2) (1965), 113. - :MULDER, E. G., and VAN VEEN, W. L.: Effect of pH and organic compounds on nitrogen fixation by red-clover. Plant and Soil 13 (2) (1960), 91. - N EWBOULD, F. H. S.: Studies on humus type legume-inoculants. Growth and survival in storage. Sci. Agri. 31 (1951), 463. - PAPADAKIS, J. S.: An important effect of soil colloids on plant growth. Soil Sci. 52 (1941), 283. - PATRICK, Z. A., and KOCH, L. W.: Inhibition of respiration, germination, and growth by substances arising during the decomposition of certain plant residues in the soil. Can. J. Bot. 36 (1958), 621. - PUGASHETTI, B.K., GOPALGOWDA, H. S., and PATH" R B.: Cellulose powder as legume inoculant base. Curro Sci. 40 (18) (1971),494. - SANKARAM, A.: Nature and quality of legume inoculant. Sci. and Culture. 25 (10) (1960), 569. - SINGH, R. S., and SITARAMAIAH, K.: Effect of decomposing green leaves, sawdust, and urea on the incidence of root-knot of okra and tomato. Indian Phytopathol. 20 (4) (1967), 349. - TINCKER, :M. A. H.: Experiments with Lilies at Wisley. VI. A summary of observation made concerning Lily roots and soil conditions. The Lily-year-book. Roy. Hort. Soc.
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London. 1947. - TURNER, E. R.: The effect of certain adsorbants on the nodulation of clover plants Ann. Bot. 19 (1955),149. - VINCENT, J.M.: Australian studies of the root nodule bacteria. Proc. Linn. Soc. N.S.W .87 (1962), 8.-VINCENT, J.M.: In "Soil-Nitrogen"; Bartholomew, W. W. and Clark, F.E. (Ed.). Amer. Soc. Agron. Monograph ch. 10 (1965),384. -ZINDZADZE, C.H.: Nutrition artificielle des plantes cultivatus Me'langes nutritifs a pH stable. Ann. Agron. Paris (N. S.) 2 (1932), 809.
Authors' address: Dr. Maitryee Pramanick and Dr. V.Iswaran, Division of Microbiology, Indian Agricultural Research Institute, New Delhi - 12 (INDIA)-