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Tissue culture bioassay for plant growth promoting rhizobacteria
Sod Bid. Eiochrm. Vol. 23. No. 4. pp.331-333. 1991 Pnntcd in Great Britain. All rights rcrer~ed
0038-0717/91 s3.00 + 0.00 copyright c
I99I Pcrgamoo Prcapk
TISSUE CULTURE BIOASSAY FOR PLANT GROWTH PROMOTING RHIZOBACTERIA* C. P. CHANWAY’~ and L. M. NEUO~*‘: ‘Department of Forest Sciences, University of British Columbia Vancouver. British Columbia, Canada V6T IWS and rNational Research Council of Canada, Plant Biotechnology Institute Saskatoon. Saskatchewan, Canada S7N 0W9 (Accepred I Nocember 1990) Summary-Plant growth promoting rhizobacteria (PGPR) Pseudomonospurida strains GZ-8 and GI l-32 were tested for growth promotion of soybean callus in a tissueculture bioassay. When grown on a nutrient medium without naphthaleneacetic acid or kinetin, PGPR did not affect callus biomass. However, when growth regulators were included in the growth medium, but vitamins (thiamine HCI. nicotinic acid, pyridoxine HCI. and myoinositol) omitted. inoculation with strain GI l-32 significantly increased thecallus biomass. Strain GZ-8 decreased the callus biomass without vitamins, but the effect was not significant in any single experiment. Results are discussed in relation to the usefulnessof tissue-culture based bioassays for PGPR and the mechanism by which PGPR stimulate plant growth.
INTRODUCTION
Plant growth promoting rhizobactcria (PGPR) may prove to be a valuable technology in agriculture (Kloeppcr CI ul., 1989) and forestry (Chanway EI al.. 1991) but not all PGPR arc equally cffcctivc in promoting plant growth (Klocppcr et crl., 1988). Diffcrcnces bctwcwn and within spccics of PGPR arc well documcntcd (Chanway CI ul., 1989). thcrcf’ore, a rapid and rcliablc assay for scrcyning putative PGPR would bc of great value in advancing this technology. To this end, root elongation assaysin growth pouches have been tested with some success(Elliot and Lynch, 1984; Lifshitz et al.. 1987). but the predictive value of these results for plant growth promotion in grcenhouse or field trials is questionable (Kloepper and Schroth. 1981; Chanway ef al., 1989). A soybean callus tissue culture bioassay has been used to effectively test for the presence of cytokinin activity in biologically-active plant extracts (Miller, 1965). We have obtained results in less than 2 weeks using this technique (Holl er al., 1988). The possibility of modifying this type of bioassay to perform rapid screening of putative PGPR has not been examined. Pseu&nonus putida strains G2-8 and Gl l-32 promote growth of agricultural crops including lentil (Lens esculenra Moench (Chanway er 01.. 1989; Dr J. Kloepper. personal communication]. The mechanism by which these PGPR stimulate plant growth is unknown, but the production of plant growth regulators by growth-promoting bacteria in general (Brown, 1974; Holl ef al.. 1988). and these strains specifically (Chanway et al.. l989), has been suggested. The objectives of this study were (i) to determine if a soybean callus bioassay could be adapted for use as a rapid screening method for PGPR. and (ii) to determine if plant growth promotion by P. putida strains G2-8 and GI l-32 involves lNRCC No. 32456. tTo whom all correspondence should be addressed.
the production of plant growth regulators or vitamins. MATERIALS AND METHODS
Cullus culture
Soybean [GIycine mux L. (Mcrr.) var. Acme] callus gcncratcd from cotyledon tissue was obtained from Mr R. Radlcy. Forest Biotechnology Centre, British Columbia Rcscarch Corporation, and was maintained on a nutrient medium based on that of Miller (1965) (Table I) at 4°C. Bucreriul struins P. putidu strains G2-8 and Gl l-32 are PGPR for agricultural crops (Chanway er al., 1989) and were obtained from Dr J. Kloepper (Allelix Inc. Mississauga, Ontario). Isolates were maintained frozen (- 80°C) in tryptic soy broth (TSB) containing 10% glycerol. Bioassay
Three media were prepared with nutrients as described in Table 1. Each medium had one of the following components (or classes of components) omitted: vitamins, naphthaleneacetic acid, or kinetin, and were designated -V. -N and -K. respectively. Medium (25 ml) was added to 250 ml flasks and autoclaved before experiments. Callus stock culture was incubated on complete nutrient medium (Table 1) at 30°C for I week before assays, and was then aseptically divided into 0.5 cm’ segments. For bioassays, two pieces of callus were placed in each flask containing hardened growth medium. P. putida was grown to stationary phase in TSB (2 days at 30°C on a rotary shaker). Bacterial cultures were harvested by centrifugation (lO.OOOg for 20 min) and washed twice in 0. I M MgSO,, before resuspending in the same buffer to IO9 colony forming units ml-t. Three replicate flasks containing pieces of callus were then inoculated with I ml of bacterial suspensions containing strain
331
c.
332
P. CHASWAY and L. M. N-N
Table I. Constituenlr of media used for soybean callus bioassay Compound
(mgl-‘1
KH,PO.
300 loo0 500
KN& Ca(NO,)+H:O Cu(NO,),-3H,O
0.35 2.45 1.9 1.6 0.8 loo0
MnSO,-H,O &SO,-7H,O %BO, KI NH,NO, EDTA(Na,kZH:O FeSO,-7H,O MgSO,-7Hz0 KCI (NH,),Mo,O:,-4H,O Thiamine HCI* Nicolinic acid* Pyridoxinc HCI* Mvoinositol* N;phthalcncacetic scidt Kinetint
13.4 9.9 71.5 65 0.1 0.8 2 0.8 loo 2 0.5
SWXOSC
308
A&T-Z
IOL!
*Omitted from -V medium. tomitted from -N medium. !Omittcd from -K medium.
GZ-8 or GI l-32. Control flasks were inoculated with 0.1 M MgSO,. After I2 days growth in the dark at 30°C. callus samples were removed from flasks, blotted dry, and wcighcd. Trcatmcnts wcrc rcpcated up to 4 times in subsequent cxpcrimcnts with bacterial inoculum or supernatant from bacterial cultures. Data were analyzed using ANOVA and means were scparatcd using Fisher’s protected LSD. RFSULTS
lation with P.put& strain Gll-32 significantly increased callus biomass on -V media (33%. P < 0.05). but strain GZ-8 decreased biomass (25%. P > 0.05). The trend for G2-8 to decrease callus biomass was reproducible, but not statistically significant in any one experiment. There is no obvious explanation for the consistent biomass reduction due to inoculation with strain G2-8 on -V medium, but competition for nutrients or the production of growth inhibitors by bacteria are possible causes of this effect. These data suggest that the mechanism by which strain GI l-32 promotes callus growth may involve the provision of vitamin-like growth factors, but not plant growth regulators similar to auxin or cytokinin. The failure of strain GZ-8 to promote callus growth in this system suggests that the mechanism of its PGPR activity is unrelated to vitamin, auxin or cytokinin production. Differences in the plant growth-promoting ability of strains GZ-8 and Gil-32 are at least in part attributable to plant cultivar effects (Chanway et al., 1989). Root growth promotion is often observed to be the initial, or sometimes the sole effect of inoculation with PGPR (Holl et ul., 1988; Chanway el 01.. 1988; Chanway et al., 1989). Therefore, to effectively screen for putative PGPR using this type of bioassay, callus derived from root tissue of the “target” plant cultivar may be most useful. It can be concluded that a tissue-culture based bioassay for PGPR may be of value, but substantial preliminary work is ncccssary to dcfinc the conditions under which PGPR activity can be dctcctcd. Further experimentation with strain GI l-32 should be undcrtaken to detcrminc if its PGPR activity is related to vitamin production in situ.
AND DISCUSSION
Preliminary work suggested that growth promotion would not be detected when bioassays were performed using the full nutrient medium. Therefore, PGPR inoculations were performed with callus growing on medium that was missing at least one important growth factor, i.e. -V, -K. or -N (Table 1). The composition of the growth medium significantly aflccted callus biomass (Table 2). Callus biomass was lowest when grown on medium without growth regulators, i.e. naphthalaneacetic acid or kinetin. This result was not unexpected because soybean callus derived from var. Acme has been previously shown to be very sensitive to the concentration of growth regulators in media, particularly kinetin (Miller, 1965; Holl ef 01.. 1988). Bacterial inoculation had no effect on callus biomass when grown on -N or -K media. Callus biomass was highest when media contained growth regulators but not vitamins (Table 2). Inocu-
REFERENCES
Brown M. E. (1974) Seed and root bacterization. Annuul Review of Phytopathology
12, 181-197.
Chanway C. P., Hynes R. K. and Nelson L. M. (1989) Plant growth promoting rhitobactcria: effects on growth and nitrogen fixation of lentil (I!XN esculennraMoench) and pea (Pisum sarivum). Soil Biology (G Biochemislry 21, 511-517.
Chanway C. P., Nelson L. M. and Hall F. 8. (1988) Cultivar specific growth promotion of spring wheat (‘friricum aesrbum) by co-existent Bacillus species. Can&an journal of Microbiology
34, 925-929.
Chanway C. P., Radley R. A. and Holl F. B. (1991) Inoculation of conifer seed with plant growth promoting Bucillus strains causes increased seedling emergence and biomass. Soil Biology d Biochemistry. In press. Elliot L. F. and Lynch J. M. (1984) Pseudomonads as a factor in the growth of winter wheat (Trilicum uesfiuum
L.).
Soil Biolonv Lb Biochemistry
16. 69-71.
Holl’F. Table 2. Effect of nucrienl medium and inoculationwith PGPR strains G2.8 or GI l-32 on mean rovbean callus biomass (mn) Medium* -V
-N
Conlrol
__...._.
279’
19lC
god
G?-a
ZIO’.’
179’ 148’.6
806 766
lnoculalion treatment
GI 1-32 *See Table
370b
I
-K
for dctiniGonr of -V. .N and -K media. Means designated with different alphabckal ruperxripcs are stalk& tally different (n - 3; LSD P < 0.05 I 78 mg).
B.. Cha&ay C. P., Turkingtdn R. and Radley R. A. (1988) Growth response of crested wheatgrass (Agropyron crisrarum L.), white clover (Tr~filium repens L.). and perennial rycgrass (Lolium perenne L.) to inoculation with Bacillus polymyxo. Soil Biology & Biochemirfry 20. 19-24.
Kloepper J. W. and Schroth M. N.
(1981)
Plant growth-
promoting rhizobactcria and plant growth under gnotobiotic conditions. Phytopalhology 71, 642-644.
Klocpper J. W., Lifshitz R. and Zablotowicz R. M. (1969) Free-living bacterial inocula for enhancing crop productivity. Trends in Biotechnology
1, 39-44.
Tissue culture bioassay for PGPR Kloepper J. W.. Hume D. J.. Schcr F. M., Singleton C.. Tipping B.. Laliberte M.. Fraulcy K.. Kutchaw T.. Simonson C.. Lifshitz R.. Zaleska 1. and Lee L. (1988) Plant growth-promoting rhizobactcria on canola (rapeseed). Plant Disease l2.42-46. Lifshitz R.. Kloepper J. W., Kozlowski M.. Simonson C.. Carlso J., Tipping E. M. and Zalaka I. (1987) Growth
promotion
333
of canola (rapeseed) by a strain of Pseuunder gnotobiotic conditions. Canadian
domotuu puti& Journal
of Microbiology
33, 3-395.
Miller C. 0. (1965) Evidence for the natural occurrence of zeatin and derivatives. Compounds from maize which promote all division. Promdings of rhc National Aca&my of Sciences, U.S.A. 54, 1052-1058.
0038-0717/91 s3.00 + 0.00 copyright c
I99I Pcrgamoo Prcapk
TISSUE CULTURE BIOASSAY FOR PLANT GROWTH PROMOTING RHIZOBACTERIA* C. P. CHANWAY’~ and L. M. NEUO~*‘: ‘Department of Forest Sciences, University of British Columbia Vancouver. British Columbia, Canada V6T IWS and rNational Research Council of Canada, Plant Biotechnology Institute Saskatoon. Saskatchewan, Canada S7N 0W9 (Accepred I Nocember 1990) Summary-Plant growth promoting rhizobacteria (PGPR) Pseudomonospurida strains GZ-8 and GI l-32 were tested for growth promotion of soybean callus in a tissueculture bioassay. When grown on a nutrient medium without naphthaleneacetic acid or kinetin, PGPR did not affect callus biomass. However, when growth regulators were included in the growth medium, but vitamins (thiamine HCI. nicotinic acid, pyridoxine HCI. and myoinositol) omitted. inoculation with strain GI l-32 significantly increased thecallus biomass. Strain GZ-8 decreased the callus biomass without vitamins, but the effect was not significant in any single experiment. Results are discussed in relation to the usefulnessof tissue-culture based bioassays for PGPR and the mechanism by which PGPR stimulate plant growth.
INTRODUCTION
Plant growth promoting rhizobactcria (PGPR) may prove to be a valuable technology in agriculture (Kloeppcr CI ul., 1989) and forestry (Chanway EI al.. 1991) but not all PGPR arc equally cffcctivc in promoting plant growth (Klocppcr et crl., 1988). Diffcrcnces bctwcwn and within spccics of PGPR arc well documcntcd (Chanway CI ul., 1989). thcrcf’ore, a rapid and rcliablc assay for scrcyning putative PGPR would bc of great value in advancing this technology. To this end, root elongation assaysin growth pouches have been tested with some success(Elliot and Lynch, 1984; Lifshitz et al.. 1987). but the predictive value of these results for plant growth promotion in grcenhouse or field trials is questionable (Kloepper and Schroth. 1981; Chanway ef al., 1989). A soybean callus tissue culture bioassay has been used to effectively test for the presence of cytokinin activity in biologically-active plant extracts (Miller, 1965). We have obtained results in less than 2 weeks using this technique (Holl er al., 1988). The possibility of modifying this type of bioassay to perform rapid screening of putative PGPR has not been examined. Pseu&nonus putida strains G2-8 and Gl l-32 promote growth of agricultural crops including lentil (Lens esculenra Moench (Chanway er 01.. 1989; Dr J. Kloepper. personal communication]. The mechanism by which these PGPR stimulate plant growth is unknown, but the production of plant growth regulators by growth-promoting bacteria in general (Brown, 1974; Holl ef al.. 1988). and these strains specifically (Chanway et al.. l989), has been suggested. The objectives of this study were (i) to determine if a soybean callus bioassay could be adapted for use as a rapid screening method for PGPR. and (ii) to determine if plant growth promotion by P. putida strains G2-8 and GI l-32 involves lNRCC No. 32456. tTo whom all correspondence should be addressed.
the production of plant growth regulators or vitamins. MATERIALS AND METHODS
Cullus culture
Soybean [GIycine mux L. (Mcrr.) var. Acme] callus gcncratcd from cotyledon tissue was obtained from Mr R. Radlcy. Forest Biotechnology Centre, British Columbia Rcscarch Corporation, and was maintained on a nutrient medium based on that of Miller (1965) (Table I) at 4°C. Bucreriul struins P. putidu strains G2-8 and Gl l-32 are PGPR for agricultural crops (Chanway er al., 1989) and were obtained from Dr J. Kloepper (Allelix Inc. Mississauga, Ontario). Isolates were maintained frozen (- 80°C) in tryptic soy broth (TSB) containing 10% glycerol. Bioassay
Three media were prepared with nutrients as described in Table 1. Each medium had one of the following components (or classes of components) omitted: vitamins, naphthaleneacetic acid, or kinetin, and were designated -V. -N and -K. respectively. Medium (25 ml) was added to 250 ml flasks and autoclaved before experiments. Callus stock culture was incubated on complete nutrient medium (Table 1) at 30°C for I week before assays, and was then aseptically divided into 0.5 cm’ segments. For bioassays, two pieces of callus were placed in each flask containing hardened growth medium. P. putida was grown to stationary phase in TSB (2 days at 30°C on a rotary shaker). Bacterial cultures were harvested by centrifugation (lO.OOOg for 20 min) and washed twice in 0. I M MgSO,, before resuspending in the same buffer to IO9 colony forming units ml-t. Three replicate flasks containing pieces of callus were then inoculated with I ml of bacterial suspensions containing strain
331
c.
332
P. CHASWAY and L. M. N-N
Table I. Constituenlr of media used for soybean callus bioassay Compound
(mgl-‘1
KH,PO.
300 loo0 500
KN& Ca(NO,)+H:O Cu(NO,),-3H,O
0.35 2.45 1.9 1.6 0.8 loo0
MnSO,-H,O &SO,-7H,O %BO, KI NH,NO, EDTA(Na,kZH:O FeSO,-7H,O MgSO,-7Hz0 KCI (NH,),Mo,O:,-4H,O Thiamine HCI* Nicolinic acid* Pyridoxinc HCI* Mvoinositol* N;phthalcncacetic scidt Kinetint
13.4 9.9 71.5 65 0.1 0.8 2 0.8 loo 2 0.5
SWXOSC
308
A&T-Z
IOL!
*Omitted from -V medium. tomitted from -N medium. !Omittcd from -K medium.
GZ-8 or GI l-32. Control flasks were inoculated with 0.1 M MgSO,. After I2 days growth in the dark at 30°C. callus samples were removed from flasks, blotted dry, and wcighcd. Trcatmcnts wcrc rcpcated up to 4 times in subsequent cxpcrimcnts with bacterial inoculum or supernatant from bacterial cultures. Data were analyzed using ANOVA and means were scparatcd using Fisher’s protected LSD. RFSULTS
lation with P.put& strain Gll-32 significantly increased callus biomass on -V media (33%. P < 0.05). but strain GZ-8 decreased biomass (25%. P > 0.05). The trend for G2-8 to decrease callus biomass was reproducible, but not statistically significant in any one experiment. There is no obvious explanation for the consistent biomass reduction due to inoculation with strain G2-8 on -V medium, but competition for nutrients or the production of growth inhibitors by bacteria are possible causes of this effect. These data suggest that the mechanism by which strain GI l-32 promotes callus growth may involve the provision of vitamin-like growth factors, but not plant growth regulators similar to auxin or cytokinin. The failure of strain GZ-8 to promote callus growth in this system suggests that the mechanism of its PGPR activity is unrelated to vitamin, auxin or cytokinin production. Differences in the plant growth-promoting ability of strains GZ-8 and Gil-32 are at least in part attributable to plant cultivar effects (Chanway et al., 1989). Root growth promotion is often observed to be the initial, or sometimes the sole effect of inoculation with PGPR (Holl et ul., 1988; Chanway el 01.. 1988; Chanway et al., 1989). Therefore, to effectively screen for putative PGPR using this type of bioassay, callus derived from root tissue of the “target” plant cultivar may be most useful. It can be concluded that a tissue-culture based bioassay for PGPR may be of value, but substantial preliminary work is ncccssary to dcfinc the conditions under which PGPR activity can be dctcctcd. Further experimentation with strain GI l-32 should be undcrtaken to detcrminc if its PGPR activity is related to vitamin production in situ.
AND DISCUSSION
Preliminary work suggested that growth promotion would not be detected when bioassays were performed using the full nutrient medium. Therefore, PGPR inoculations were performed with callus growing on medium that was missing at least one important growth factor, i.e. -V, -K. or -N (Table 1). The composition of the growth medium significantly aflccted callus biomass (Table 2). Callus biomass was lowest when grown on medium without growth regulators, i.e. naphthalaneacetic acid or kinetin. This result was not unexpected because soybean callus derived from var. Acme has been previously shown to be very sensitive to the concentration of growth regulators in media, particularly kinetin (Miller, 1965; Holl ef 01.. 1988). Bacterial inoculation had no effect on callus biomass when grown on -N or -K media. Callus biomass was highest when media contained growth regulators but not vitamins (Table 2). Inocu-
REFERENCES
Brown M. E. (1974) Seed and root bacterization. Annuul Review of Phytopathology
12, 181-197.
Chanway C. P., Hynes R. K. and Nelson L. M. (1989) Plant growth promoting rhitobactcria: effects on growth and nitrogen fixation of lentil (I!XN esculennraMoench) and pea (Pisum sarivum). Soil Biology (G Biochemislry 21, 511-517.
Chanway C. P., Nelson L. M. and Hall F. 8. (1988) Cultivar specific growth promotion of spring wheat (‘friricum aesrbum) by co-existent Bacillus species. Can&an journal of Microbiology
34, 925-929.
Chanway C. P., Radley R. A. and Holl F. B. (1991) Inoculation of conifer seed with plant growth promoting Bucillus strains causes increased seedling emergence and biomass. Soil Biology d Biochemistry. In press. Elliot L. F. and Lynch J. M. (1984) Pseudomonads as a factor in the growth of winter wheat (Trilicum uesfiuum
L.).
Soil Biolonv Lb Biochemistry
16. 69-71.
Holl’F. Table 2. Effect of nucrienl medium and inoculationwith PGPR strains G2.8 or GI l-32 on mean rovbean callus biomass (mn) Medium* -V
-N
Conlrol
__...._.
279’
19lC
god
G?-a
ZIO’.’
179’ 148’.6
806 766
lnoculalion treatment
GI 1-32 *See Table
370b
I
-K
for dctiniGonr of -V. .N and -K media. Means designated with different alphabckal ruperxripcs are stalk& tally different (n - 3; LSD P < 0.05 I 78 mg).
B.. Cha&ay C. P., Turkingtdn R. and Radley R. A. (1988) Growth response of crested wheatgrass (Agropyron crisrarum L.), white clover (Tr~filium repens L.). and perennial rycgrass (Lolium perenne L.) to inoculation with Bacillus polymyxo. Soil Biology & Biochemirfry 20. 19-24.
Kloepper J. W. and Schroth M. N.
(1981)
Plant growth-
promoting rhizobactcria and plant growth under gnotobiotic conditions. Phytopalhology 71, 642-644.
Klocpper J. W., Lifshitz R. and Zablotowicz R. M. (1969) Free-living bacterial inocula for enhancing crop productivity. Trends in Biotechnology
1, 39-44.
Tissue culture bioassay for PGPR Kloepper J. W.. Hume D. J.. Schcr F. M., Singleton C.. Tipping B.. Laliberte M.. Fraulcy K.. Kutchaw T.. Simonson C.. Lifshitz R.. Zaleska 1. and Lee L. (1988) Plant growth-promoting rhizobactcria on canola (rapeseed). Plant Disease l2.42-46. Lifshitz R.. Kloepper J. W., Kozlowski M.. Simonson C.. Carlso J., Tipping E. M. and Zalaka I. (1987) Growth
promotion
333
of canola (rapeseed) by a strain of Pseuunder gnotobiotic conditions. Canadian
domotuu puti& Journal
of Microbiology
33, 3-395.
Miller C. 0. (1965) Evidence for the natural occurrence of zeatin and derivatives. Compounds from maize which promote all division. Promdings of rhc National Aca&my of Sciences, U.S.A. 54, 1052-1058.