- Email: [email protected]
Studies on the rhizosphere microflora of onion plants in relation to temperature changes
Soil Bid. Biochem. Vol. 5, pp. 315-320. Pergamon Press 1973. Printed in Great Britain.
STUDIES ON THE RHIZOSPHERE MICROFLORA OF ONION PLANTS IN RELATION TO TEMPERATURE CHANGES LYNDSAY FENWICK* Department
of Botany, University of Canterbury, (Accepted
Christchurch,
New Zealand
5 October 1972)
Summary-A study was made of the changes in the rhizosphere microflora of onion seedlings grown under controlled conditions at either 25, 18 or 16°C. In plants grown at the lower temperatures there was a very small rhizosphere effect for the bacteria compared with that for plants grown at 25°C. A series of tests on bacteria isolated from both rhizosphere and root-free soil showed the rhizosphere isolates from seedlings grown at 25°C to be more active physiologically than those from seedlings grown at the lower temperatures. In particular, the ammonifying bacteria were greatly increased in the rhizosphere of plants grown at 25°C. Numbers of rhizosphere fungi were also greater at the higher temperature but the rhizosphere effect was not as great as that for bacteria. Contrary to expectation the greatest rhizosphere effect did not occur at the optimum temperature for growth of the onions. INTRODUCTION
of various plant species have shown that groups of microorganisms occur with differing frequencies and that their numbers are greatly influenced both directly and indirectly by the age of the plants sampled, the nature and treatment of the soil and the environmental conditions under which the plants are grown (Rovira, 1965). Because of the importance of environmental factors a study has been made of the rhizosphere of onion seedlings grown under controlled conditions. Results from two previous reports (Timonin and Thexton, 1951; Strzelczyk, 1961) vary widely and it may be assumed that this is due to the different and largely uncontrolled conditions under which the plants were grown and to the difference in ages when the rhizospheres were sampled. Temperature is one of the environmental factors affecting the rhizosphere which has received particular attention. Rouatt et al. (1963) have demonstrated specific changes in the rhizosphere microbial populations of wheat and soybean plants grown at different temperatures and have shown these changes to be greater than the effect produced by the direct action of temperature on the soil. Environmental conditions can affect the nature of root exudates (Rovira, 1959) and it is in this way that variations in temperature modify to a large extent the rhizosphere flora. In the present study the rhizosphere of onion seedlings was examined with particular reference to the variations in microbial populations with changes in the temperature at which the plants were grown. For comparison the temperatures were chosen within the normal range for growth of onions, and above this. COMPARISONS between
the rhizospheres
MATERIALS
AND METHODS
Plant and soil techniques Onion seeds (Allium cepa var. Pukekohe Long Keeper) were surface sterilized by immersion in 0 * 1 ‘A (w/v) solutions of streptomycin sulphate and mercuric chloride and were * Present address: 17 Mainston Road, Remuera, Auckland 5, New Zealand. 315
316
LYNIXAY FENWICK
germinated and grown on malt extract agar for 7 days at either 20 or 25°C depending on subsequent treatment. The seeds were sterilized in this way to eliminate the introduction of seed-coat contaminants into the soil. Those which were free from contamination were grown in soil contained in 3 x 20 cm test tubes, three to a tube. All soil was taken from a bulk supply of air-dried sandy loam with added humus which had been sieved through a 5 mm mesh. A washed glass wool pad was placed in the bottom of each tube before 40 g of soil was added. The moisture level of the soil was brought to 60 per cent water-holding capacity (Piper, 1950) by the addition of IO-2 ml of water, and the seedlings were then planted. The tubes were capped with aluminium foil to reduce moisture loss. The onion seedfings were grown for 21-27 days before sampIing the rhizosphere. During this time the temperature was maintained at either 25 riT:2°C 18 rf 2°C or 16 + 1°C and the seedlings were grown with 12 h of light at an intensity of 6000 lx in each 24-h period. Sampling techniques Seedlings were carefully removed from the tubes and shaken to dislodge the looselyadhering soil (Timonin, 1940). The roots from four seedlings were used for each sample and these were shaken for 2 min in 100 ml of sterile tap water (Loutit and Loutit, 1966). The roots were removed and 10 g of glass beads (3 mm diam.) were added to the suspension which was shaken for 2 min. The container was allowed to stand for 30 s before preparing a tenfold dilution series. All the dilutions were shaken in the same manner with the inclusion of glass beads (Brown et al., 1963). Since Loutit (1966) suggested that there was less variation in the numbers of microorganisms counted from small quantities of rhizosphere soil than from larger amounts, the weight of oven-dried rhizosphere soil from onion roots was maintained between 30 and 50 mg. Five grams of root-free soil was sampled as a control from the tubes in which the seedlings were grown. Four 1 ml replicates from each dilution were plated on Martin’s (1950) medium selective for fungi and Bunt and Rovira’s (1955) soil extract medium selective for bacteria. The eubacteria and the actinomycetes were counted as one group. Samples from the plants grown at 16 and 18°C were incubated at 20°C while those from plants grown at 25°C were incubated at 25°C. The numbers of bacterial colonies developing were counted after 14 days and fungi after 4 and 7 days.
Bacterial plates with 20-40 colonies were selected and all colonies were subcultured on Bunt and Rovira agar plates. Physiological tests (Katznelson and Rouatt, 1957) were used to group these isolates. The tests included the ability to reduce methylene blue, production of acid and gas from a glucose medium, denitrification and ammonification of specific media. Bunt and Rovira’s liquid medium containing 0.1 per cent glucose was used for methylene blue reduction, acid and gas production tests, the cultures being examined at intervals of 2 or 3 days for a fortnight. The presence of ammonifying and denitrifying bacteria was tested 7 and 12 days after inoculation of the appropriate media. The fungi were classified into broad generic groups from direct observation of the dilution plates or by subculturing colonies on to malt extract agar, followed by microscopic examination. Two series of onion seedlings were grown at 16”C, one at 18°C and two at 25°C. There were three replicates of each series and the numbers of bacteria and fungi per gram of ovendried soil in rhizosphere and root-free soil were determined. Throughout the experiment care was taken to prevent wounding of the onion roots and thus reduce the production of thiolsulphanates which Virtanen and Matikkala (1959)
TEMPERATURE
AND ONION
RHIZOSPHERE
317
MICROFLORA
showed to have strong antimicrobial effects. It was found from a trial made with the first rhizosphere sample from seedlings grown at 25°C that shaking the roots with glass beads did in fact reduce the count of microorganisms so that in all subsequent experiments roots were shaken in sterile water to dislodge the adhering soil and microorganisms, and then removed before the glass beads were added. RESULTS
When the microfloras from the root-free soil at the two temperatures were compared there were signi~~antly more bacteria at 16°C than at 25°C while the reverse was true of fungi (Tables 1 and 2). TABLE
1. NUMBERS
Temperature Age . ..
OF BACTERIA (106/g OVEN-DRY SOIL)IN THE RHIZO~PHERE SOILIN RELATIONTOTEMPERATURE
...
_..
. ..
. _.
Rhizosphere Soil R:S
16 & 1°C 22 days 7.6 rfI 0.9* 18.9 & 1.0 0.40:1
16 _L 1°C 27 days 28-5 & 3.3 19.2 + 0.9 1.48:1
0~ ONIONS AND
TWE ROOT-FREE
18 i 2°C 21 days
25 * 2°C 21 days
25 * 2°C 21 days
16.4 f 1.5 15.1 * 1.2 1.08:1
121.4 f 25.3 12.0 f 1.4 10.12: 1
110.0 f 12.1 16.0 f 1.4 6.88: 1
* Each value represents the mean of 12 samples together with the standard error of the mean.
TABLE 2. NUMBERS
Temperature Age .. . Rhizosphere Soil R:S
OF FUNGI(~O~/~ OVEN-DRY SOIL)IN THE RHIZOSPHERE OF ONIONS AND THE ROOT-FREE SOIL INRELATIONTOTEMPERATURE
.. .
.. .
. ..
...
16 I;t: 1°C 22 days
16 & 1°C 27 days
18 f 2°C 21 days
6-l It: 0.4* 3.2 ic 0.3 1.91:1
14.9 * 1.2 8.0 zt 0.7 1.86:1
20.1 i f-4 6.8 + 0.3 2.96: 1
25 & 2°C 21 days 80.1 rt 9.0 15.2 f 2.5 5.27:1
25 i 2°C 21 days 53.4 i 4.2 10.3 f 0.2 5.18:1
* Each value represents the mean of 12 samples together with the standard error of the mean.
The numbers of bacteria in the rhizosphere of plants grown at 16 and 18°C compared with root-free soil varied markedly over the three series of experiments. For plants 22 days old and grown at 16°C the R: S ratio was 0.40 showing that there were significantly fewer bacteria in the rhizosphere than in root-free soil. In the second experiment where plants were grown at 18°C for 21 days there were only slightly more bacteria in the rhizosphere than in root-free soil and there was no significant difference between the numbers. A rhizosphere effect was more evident in plants a week older, grown at 16”C, although the R: S ratio was only l-48. The R: S ratios for fungi occurring in plants grown at the lower temperature were similar to those for bacteria in being rather small. They varied from 1.86 to 2.96 but in all cases there were significantly more in the rhizosphere than in root-free soil. For seedlings grown at 25°C the R: S ratios for bacteria were seven to ten times higher than the ratios for seedlings grown at the lower temperatures and the ratios for fungi were also greatly increased. While the effect of root-free soil was to induce more bacterial growth at 16 and 18°C than at the higher temperature, the rhizosphere of the onion seedlings reversed this.
318
LYNDSAY
FENWICK
From the first experiment at 25”C, 50 rhizosphere and 50 root-free soil bacteria were isolated and subcultured on to Bunt and Rovira’s agar. Six rhizosphere and two root-free soil isolates failed to grow. Fifty isolates from rhizosphere and root-free soil dilutions of plants grown at 16°C for 22 days were made but over half of these failed to grow. A further 31 isolates from both rhizosphere and root-free soil plates of plants grown for 27 days at 16°C were made. Because of variations in age of the seedlings in the two series sampled at 16°C and the R: S ratio differences, results from these series have been recorded separately (Table 3).
TABLE 3. NUMBERS
Temperature
Physiological
OF BACTERIA(~~~/~ OVEN-DRY SOIL)IN VARIOUS PHYSIOLOGICAL GROUPS IN RELATION TO TEMPERATURE
. group
Methylene blue reducers Acid from glucose Gas from glucose Ammonifiers Denitrifiers
.. . . .
16°C (first series) . ..
R
S
R: S
3.38 4.06 0 5.41 5.41
7.58 2.84 0 8.52 5.68
0.45 1.43 0 0.64 0.95
16°C (second series) R 12.88 7.36 0 10.12 11.96
S IO.22 4.47 0 8.95 3.83
R:S 1.26 1,65 0 1.13 3.12
25°C R 46.92 55.20 0 77.28 19.32
S
R:S
5.25 4.50 0.75 4.00 2.00
9.96 12.27 19.32 9.66
There were fewer methylene blue reducers, ammonifiers and denitrifiers in root-free soil at 25°C than at 16°C while there was a slight increase in acid and gas producers (Table 3). Despite the differences between the two groups of bacteria from plants grown at 16°C the R: S ratios were significantly sma!ler than those where the plants were grown at 25°C. Although the soil bacteria were not as active at the higher temperature the onion seedlings stimulated more physiologically active groups at this temperature than at 16°C. There were greater numbers of bacteria in all the physiological groups except the gas producers in the rhizospheres of plants grown at 25°C but the ammonifiers responded with the greatest increases. Although the physiological tests cover a wide range of activities they cannot be regarded as independent. Statistical tests showed the groups to be linked in at least two-way combinations. This suggests that if a bacterium is physiologically active it will give positive results in two and possibly more of the tests. Counts of readily identifiable fungi were made from a large proportion of the dilution plates. Because the dilution plate method was used for their isolation, the predominant species recorded were the heavily-sporing fungi. The main genera occurring at all temperatures were Penicillium, Aspergillus, Trichoderrna and Cladosporium. The numbers of certain fungi, in particular Trichoderma and Fusarium, varied with temperature, when they were more prevalent at 25°C than at 16°C. Little could be noted for the R: S ratios of the fungi except that Penicillium was greatly stimulated in the rhizosphere of seedlings grown at 25°C giving a value of 365.5 as compared with 10.2 for seedlings grown at 18°C. The numbers of Fusarium colonies occurring in the rhizosphere of seedlings grown at 25°C was also greater than at 16”C, the R: S ratios in this case being 32.6 and 2.1 respectively.
TEMPERATURE
AND ONION
RHIZOSPHERE
MICROFLORA
319
DISCUSSION
The results show that temperature exerts a greater effect on the rhizosphere microflora of onion seedlings than can be ascribed to its effect on soil alone, indicating that the nature of the root exudates is influenced by the temperature at which seedlings are grown. Rouatt et al. (1963) showed that the numbers of bacteria occurring in the rhizosphere of plants grown at different temperatures could be equated with the optimal growth conditions for these plants. Thus the R: S ratio for wheat was highest at the lowest temperature, and that for soybeans at the highest temperature. Onions have been classed as ‘medium temperature’ plants (Mr. H. J. Giesen, personal communication). Under normal conditions the variety Pukekohe Long Keeper is planted in cool conditions (maximum soil temperature 18°C) but warmer temperatures and long days are necessary for bulb formation (Jones and Mann, 1963). The seedlings in this study had formed longer roots and were healthier at 16°C than at 25°C but the greater rhizosphere effect occurred in plants grown at the higher temperature where, in addition, the bacteria were more active physiologically. In particular there was a sevenfold increase in the R: S ratios of methylene blue reducers and acid producers at the higher temperature compared with those for seedlings grown at 16°C while there was a 17-fold increase for ammonifiers. In this case the maximum rhizosphere effect does not appear to be associated with optimum temperatures for normal growth. For the microflora of root-free soil sampled at different temperatures the results were the reverse of those reported by Rouatt et al. (1963). In the present work there were fewer bacteria in the soil at 25°C than at 16°C and the physiological activity was lower at the higher temperature. However, the present comparison was made over a smaller temperature range and the light and moisture conditions probably differed. The two previous rhizosphere studies of onions differ from each other and from the present work. With varying soil conditions Strzelczyk (1961) reported bacterial R:S ratios of 3 -7 and 5 -8 for seedlings 3 weeks old, these values lying between the ratios for seedlings grown at different temperatures in the present work. The physiological activity of the isolates varied widely with differing soil conditions apart from gas production which was uniformly low. Timonin and Thexton’s (1951) plants were sampled at 117 days and gave R: S ratios of 11.16 and 2.59 for bacteria and fungi respectively. The almost complete absence of gas-producing bacteria in rhizosphere and root-free soil is of note in the present work. Katznelson and Rouatt (1957) showed there was a large increase in gas-producers in the rhizosphere of oats and barley while Rouatt et al. (1963) reported a larger rhizosphere effect on these organisms for wheat than Strzelczyk (1961). The lack of gas-producers in the rhizosphere of onions would therefore appear to be a characteristic of these plants. Acknowledgements-This work was carried out at the University of Canterbury for the Plant Diseases Division of the D.S.I.R. The author is indebted to Dr J. D. ALLEN for his helpful guidance and to Professor W. R. PHILIPSONfor making available the facilities of the Botany Department.
REFERENCES BROWN M. E., BUUNGHAM S. K. and JACKSONR. M. (1962) Studies on Azotobacter species in soil-I. Comparison of media and techniques for counting Azotobacter in soil. PI. Soil 17, 309-319. BUNTJ. S. and ROVIRAA. D. (1955) Microbiological studies of some subantarctic soils. J. SoilSci. 6,119-128. JONESH. A. and MANN L. K. (1963) Onions and their Allies. Botany, Cultivation and Utilization. Leonard Hill, London. KATZNELXINH. and ROUATTJ. W. (1957) Studies on the incidence of certain physiological groups of bacteria in the rhizosphere. Can. J. Microbial. 3, 265-269. Loum M. (1966) The effect of microorganisms on the availability of trace elements to plants. Ph.D. thesis, University of Otago.
320
LYNDSAY
FENWICK
LOUTITM. and LoUTIT J. S. (1966) The aerobic heterotrophic bacteria of two New Zealand soils. N.Z. J. agric. Res. 9, 84-92. MARTINJ. P. (1950) Use of acid, rose bengal and streptomycin in the plate method for estimating soil fungi. Soil Sci. 69, 215-232. F?PERC. S. (1950) Soil and Plant Analysis. Waite Agricultural Research Institute, University of Adelaide. ROUATTJ. W., PETERSON E. A., KATZNELSON H. and HENDERSONV. E. (1963) Microorganisms in the root zone in relation to temperature. Can. J. Microbial. 9, 227-236. ROVIRAA. D. (1959) Root excretions in relation to the rhizosphere effect-IV. Influence of plant species, age of plant, light, temperature and calcium nutrition on exudation. PI. Soil 11,53-64. ROVIRA A. D. (1965) Interactions between plant roots and soil microorganisms. A. Rev. Microbial. 19, 241-266. STRZELCZYKE. (1961) Studies on the incidence of certain “nutritional” and physiological groups of bacteria in rhizosphere and non-rhizosphere soil. Acta Microbial. Polon. 10, 169-180. TIMONJN M. I. (1940) The interaction of higher plants and soil microorganisms-I. Microbial population of rhizosphere seedlings of certain cultivated plants. Can. J. Res. C18, 307-317. TIMONINM. I. and THEXTON R. H. (1951) The rhizosphere effect of onion and garlic on the soil microflora. Soil Sci. Sot. Am. Proc. 15,186-189. VIRTANENA. I. and MATIKKALAE. J. (1959) The isolation of S-methylcysteinesulphoxide and S-n-propylcysteinesulphoxide from onion (Allium cepa) and the antibiotic activity of crushed onion. Actu Chem. Stand. 13, 1898-1900.
STUDIES ON THE RHIZOSPHERE MICROFLORA OF ONION PLANTS IN RELATION TO TEMPERATURE CHANGES LYNDSAY FENWICK* Department
of Botany, University of Canterbury, (Accepted
Christchurch,
New Zealand
5 October 1972)
Summary-A study was made of the changes in the rhizosphere microflora of onion seedlings grown under controlled conditions at either 25, 18 or 16°C. In plants grown at the lower temperatures there was a very small rhizosphere effect for the bacteria compared with that for plants grown at 25°C. A series of tests on bacteria isolated from both rhizosphere and root-free soil showed the rhizosphere isolates from seedlings grown at 25°C to be more active physiologically than those from seedlings grown at the lower temperatures. In particular, the ammonifying bacteria were greatly increased in the rhizosphere of plants grown at 25°C. Numbers of rhizosphere fungi were also greater at the higher temperature but the rhizosphere effect was not as great as that for bacteria. Contrary to expectation the greatest rhizosphere effect did not occur at the optimum temperature for growth of the onions. INTRODUCTION
of various plant species have shown that groups of microorganisms occur with differing frequencies and that their numbers are greatly influenced both directly and indirectly by the age of the plants sampled, the nature and treatment of the soil and the environmental conditions under which the plants are grown (Rovira, 1965). Because of the importance of environmental factors a study has been made of the rhizosphere of onion seedlings grown under controlled conditions. Results from two previous reports (Timonin and Thexton, 1951; Strzelczyk, 1961) vary widely and it may be assumed that this is due to the different and largely uncontrolled conditions under which the plants were grown and to the difference in ages when the rhizospheres were sampled. Temperature is one of the environmental factors affecting the rhizosphere which has received particular attention. Rouatt et al. (1963) have demonstrated specific changes in the rhizosphere microbial populations of wheat and soybean plants grown at different temperatures and have shown these changes to be greater than the effect produced by the direct action of temperature on the soil. Environmental conditions can affect the nature of root exudates (Rovira, 1959) and it is in this way that variations in temperature modify to a large extent the rhizosphere flora. In the present study the rhizosphere of onion seedlings was examined with particular reference to the variations in microbial populations with changes in the temperature at which the plants were grown. For comparison the temperatures were chosen within the normal range for growth of onions, and above this. COMPARISONS between
the rhizospheres
MATERIALS
AND METHODS
Plant and soil techniques Onion seeds (Allium cepa var. Pukekohe Long Keeper) were surface sterilized by immersion in 0 * 1 ‘A (w/v) solutions of streptomycin sulphate and mercuric chloride and were * Present address: 17 Mainston Road, Remuera, Auckland 5, New Zealand. 315
316
LYNIXAY FENWICK
germinated and grown on malt extract agar for 7 days at either 20 or 25°C depending on subsequent treatment. The seeds were sterilized in this way to eliminate the introduction of seed-coat contaminants into the soil. Those which were free from contamination were grown in soil contained in 3 x 20 cm test tubes, three to a tube. All soil was taken from a bulk supply of air-dried sandy loam with added humus which had been sieved through a 5 mm mesh. A washed glass wool pad was placed in the bottom of each tube before 40 g of soil was added. The moisture level of the soil was brought to 60 per cent water-holding capacity (Piper, 1950) by the addition of IO-2 ml of water, and the seedlings were then planted. The tubes were capped with aluminium foil to reduce moisture loss. The onion seedfings were grown for 21-27 days before sampIing the rhizosphere. During this time the temperature was maintained at either 25 riT:2°C 18 rf 2°C or 16 + 1°C and the seedlings were grown with 12 h of light at an intensity of 6000 lx in each 24-h period. Sampling techniques Seedlings were carefully removed from the tubes and shaken to dislodge the looselyadhering soil (Timonin, 1940). The roots from four seedlings were used for each sample and these were shaken for 2 min in 100 ml of sterile tap water (Loutit and Loutit, 1966). The roots were removed and 10 g of glass beads (3 mm diam.) were added to the suspension which was shaken for 2 min. The container was allowed to stand for 30 s before preparing a tenfold dilution series. All the dilutions were shaken in the same manner with the inclusion of glass beads (Brown et al., 1963). Since Loutit (1966) suggested that there was less variation in the numbers of microorganisms counted from small quantities of rhizosphere soil than from larger amounts, the weight of oven-dried rhizosphere soil from onion roots was maintained between 30 and 50 mg. Five grams of root-free soil was sampled as a control from the tubes in which the seedlings were grown. Four 1 ml replicates from each dilution were plated on Martin’s (1950) medium selective for fungi and Bunt and Rovira’s (1955) soil extract medium selective for bacteria. The eubacteria and the actinomycetes were counted as one group. Samples from the plants grown at 16 and 18°C were incubated at 20°C while those from plants grown at 25°C were incubated at 25°C. The numbers of bacterial colonies developing were counted after 14 days and fungi after 4 and 7 days.
Bacterial plates with 20-40 colonies were selected and all colonies were subcultured on Bunt and Rovira agar plates. Physiological tests (Katznelson and Rouatt, 1957) were used to group these isolates. The tests included the ability to reduce methylene blue, production of acid and gas from a glucose medium, denitrification and ammonification of specific media. Bunt and Rovira’s liquid medium containing 0.1 per cent glucose was used for methylene blue reduction, acid and gas production tests, the cultures being examined at intervals of 2 or 3 days for a fortnight. The presence of ammonifying and denitrifying bacteria was tested 7 and 12 days after inoculation of the appropriate media. The fungi were classified into broad generic groups from direct observation of the dilution plates or by subculturing colonies on to malt extract agar, followed by microscopic examination. Two series of onion seedlings were grown at 16”C, one at 18°C and two at 25°C. There were three replicates of each series and the numbers of bacteria and fungi per gram of ovendried soil in rhizosphere and root-free soil were determined. Throughout the experiment care was taken to prevent wounding of the onion roots and thus reduce the production of thiolsulphanates which Virtanen and Matikkala (1959)
TEMPERATURE
AND ONION
RHIZOSPHERE
317
MICROFLORA
showed to have strong antimicrobial effects. It was found from a trial made with the first rhizosphere sample from seedlings grown at 25°C that shaking the roots with glass beads did in fact reduce the count of microorganisms so that in all subsequent experiments roots were shaken in sterile water to dislodge the adhering soil and microorganisms, and then removed before the glass beads were added. RESULTS
When the microfloras from the root-free soil at the two temperatures were compared there were signi~~antly more bacteria at 16°C than at 25°C while the reverse was true of fungi (Tables 1 and 2). TABLE
1. NUMBERS
Temperature Age . ..
OF BACTERIA (106/g OVEN-DRY SOIL)IN THE RHIZO~PHERE SOILIN RELATIONTOTEMPERATURE
...
_..
. ..
. _.
Rhizosphere Soil R:S
16 & 1°C 22 days 7.6 rfI 0.9* 18.9 & 1.0 0.40:1
16 _L 1°C 27 days 28-5 & 3.3 19.2 + 0.9 1.48:1
0~ ONIONS AND
TWE ROOT-FREE
18 i 2°C 21 days
25 * 2°C 21 days
25 * 2°C 21 days
16.4 f 1.5 15.1 * 1.2 1.08:1
121.4 f 25.3 12.0 f 1.4 10.12: 1
110.0 f 12.1 16.0 f 1.4 6.88: 1
* Each value represents the mean of 12 samples together with the standard error of the mean.
TABLE 2. NUMBERS
Temperature Age .. . Rhizosphere Soil R:S
OF FUNGI(~O~/~ OVEN-DRY SOIL)IN THE RHIZOSPHERE OF ONIONS AND THE ROOT-FREE SOIL INRELATIONTOTEMPERATURE
.. .
.. .
. ..
...
16 I;t: 1°C 22 days
16 & 1°C 27 days
18 f 2°C 21 days
6-l It: 0.4* 3.2 ic 0.3 1.91:1
14.9 * 1.2 8.0 zt 0.7 1.86:1
20.1 i f-4 6.8 + 0.3 2.96: 1
25 & 2°C 21 days 80.1 rt 9.0 15.2 f 2.5 5.27:1
25 i 2°C 21 days 53.4 i 4.2 10.3 f 0.2 5.18:1
* Each value represents the mean of 12 samples together with the standard error of the mean.
The numbers of bacteria in the rhizosphere of plants grown at 16 and 18°C compared with root-free soil varied markedly over the three series of experiments. For plants 22 days old and grown at 16°C the R: S ratio was 0.40 showing that there were significantly fewer bacteria in the rhizosphere than in root-free soil. In the second experiment where plants were grown at 18°C for 21 days there were only slightly more bacteria in the rhizosphere than in root-free soil and there was no significant difference between the numbers. A rhizosphere effect was more evident in plants a week older, grown at 16”C, although the R: S ratio was only l-48. The R: S ratios for fungi occurring in plants grown at the lower temperature were similar to those for bacteria in being rather small. They varied from 1.86 to 2.96 but in all cases there were significantly more in the rhizosphere than in root-free soil. For seedlings grown at 25°C the R: S ratios for bacteria were seven to ten times higher than the ratios for seedlings grown at the lower temperatures and the ratios for fungi were also greatly increased. While the effect of root-free soil was to induce more bacterial growth at 16 and 18°C than at the higher temperature, the rhizosphere of the onion seedlings reversed this.
318
LYNDSAY
FENWICK
From the first experiment at 25”C, 50 rhizosphere and 50 root-free soil bacteria were isolated and subcultured on to Bunt and Rovira’s agar. Six rhizosphere and two root-free soil isolates failed to grow. Fifty isolates from rhizosphere and root-free soil dilutions of plants grown at 16°C for 22 days were made but over half of these failed to grow. A further 31 isolates from both rhizosphere and root-free soil plates of plants grown for 27 days at 16°C were made. Because of variations in age of the seedlings in the two series sampled at 16°C and the R: S ratio differences, results from these series have been recorded separately (Table 3).
TABLE 3. NUMBERS
Temperature
Physiological
OF BACTERIA(~~~/~ OVEN-DRY SOIL)IN VARIOUS PHYSIOLOGICAL GROUPS IN RELATION TO TEMPERATURE
. group
Methylene blue reducers Acid from glucose Gas from glucose Ammonifiers Denitrifiers
.. . . .
16°C (first series) . ..
R
S
R: S
3.38 4.06 0 5.41 5.41
7.58 2.84 0 8.52 5.68
0.45 1.43 0 0.64 0.95
16°C (second series) R 12.88 7.36 0 10.12 11.96
S IO.22 4.47 0 8.95 3.83
R:S 1.26 1,65 0 1.13 3.12
25°C R 46.92 55.20 0 77.28 19.32
S
R:S
5.25 4.50 0.75 4.00 2.00
9.96 12.27 19.32 9.66
There were fewer methylene blue reducers, ammonifiers and denitrifiers in root-free soil at 25°C than at 16°C while there was a slight increase in acid and gas producers (Table 3). Despite the differences between the two groups of bacteria from plants grown at 16°C the R: S ratios were significantly sma!ler than those where the plants were grown at 25°C. Although the soil bacteria were not as active at the higher temperature the onion seedlings stimulated more physiologically active groups at this temperature than at 16°C. There were greater numbers of bacteria in all the physiological groups except the gas producers in the rhizospheres of plants grown at 25°C but the ammonifiers responded with the greatest increases. Although the physiological tests cover a wide range of activities they cannot be regarded as independent. Statistical tests showed the groups to be linked in at least two-way combinations. This suggests that if a bacterium is physiologically active it will give positive results in two and possibly more of the tests. Counts of readily identifiable fungi were made from a large proportion of the dilution plates. Because the dilution plate method was used for their isolation, the predominant species recorded were the heavily-sporing fungi. The main genera occurring at all temperatures were Penicillium, Aspergillus, Trichoderrna and Cladosporium. The numbers of certain fungi, in particular Trichoderma and Fusarium, varied with temperature, when they were more prevalent at 25°C than at 16°C. Little could be noted for the R: S ratios of the fungi except that Penicillium was greatly stimulated in the rhizosphere of seedlings grown at 25°C giving a value of 365.5 as compared with 10.2 for seedlings grown at 18°C. The numbers of Fusarium colonies occurring in the rhizosphere of seedlings grown at 25°C was also greater than at 16”C, the R: S ratios in this case being 32.6 and 2.1 respectively.
TEMPERATURE
AND ONION
RHIZOSPHERE
MICROFLORA
319
DISCUSSION
The results show that temperature exerts a greater effect on the rhizosphere microflora of onion seedlings than can be ascribed to its effect on soil alone, indicating that the nature of the root exudates is influenced by the temperature at which seedlings are grown. Rouatt et al. (1963) showed that the numbers of bacteria occurring in the rhizosphere of plants grown at different temperatures could be equated with the optimal growth conditions for these plants. Thus the R: S ratio for wheat was highest at the lowest temperature, and that for soybeans at the highest temperature. Onions have been classed as ‘medium temperature’ plants (Mr. H. J. Giesen, personal communication). Under normal conditions the variety Pukekohe Long Keeper is planted in cool conditions (maximum soil temperature 18°C) but warmer temperatures and long days are necessary for bulb formation (Jones and Mann, 1963). The seedlings in this study had formed longer roots and were healthier at 16°C than at 25°C but the greater rhizosphere effect occurred in plants grown at the higher temperature where, in addition, the bacteria were more active physiologically. In particular there was a sevenfold increase in the R: S ratios of methylene blue reducers and acid producers at the higher temperature compared with those for seedlings grown at 16°C while there was a 17-fold increase for ammonifiers. In this case the maximum rhizosphere effect does not appear to be associated with optimum temperatures for normal growth. For the microflora of root-free soil sampled at different temperatures the results were the reverse of those reported by Rouatt et al. (1963). In the present work there were fewer bacteria in the soil at 25°C than at 16°C and the physiological activity was lower at the higher temperature. However, the present comparison was made over a smaller temperature range and the light and moisture conditions probably differed. The two previous rhizosphere studies of onions differ from each other and from the present work. With varying soil conditions Strzelczyk (1961) reported bacterial R:S ratios of 3 -7 and 5 -8 for seedlings 3 weeks old, these values lying between the ratios for seedlings grown at different temperatures in the present work. The physiological activity of the isolates varied widely with differing soil conditions apart from gas production which was uniformly low. Timonin and Thexton’s (1951) plants were sampled at 117 days and gave R: S ratios of 11.16 and 2.59 for bacteria and fungi respectively. The almost complete absence of gas-producing bacteria in rhizosphere and root-free soil is of note in the present work. Katznelson and Rouatt (1957) showed there was a large increase in gas-producers in the rhizosphere of oats and barley while Rouatt et al. (1963) reported a larger rhizosphere effect on these organisms for wheat than Strzelczyk (1961). The lack of gas-producers in the rhizosphere of onions would therefore appear to be a characteristic of these plants. Acknowledgements-This work was carried out at the University of Canterbury for the Plant Diseases Division of the D.S.I.R. The author is indebted to Dr J. D. ALLEN for his helpful guidance and to Professor W. R. PHILIPSONfor making available the facilities of the Botany Department.
REFERENCES BROWN M. E., BUUNGHAM S. K. and JACKSONR. M. (1962) Studies on Azotobacter species in soil-I. Comparison of media and techniques for counting Azotobacter in soil. PI. Soil 17, 309-319. BUNTJ. S. and ROVIRAA. D. (1955) Microbiological studies of some subantarctic soils. J. SoilSci. 6,119-128. JONESH. A. and MANN L. K. (1963) Onions and their Allies. Botany, Cultivation and Utilization. Leonard Hill, London. KATZNELXINH. and ROUATTJ. W. (1957) Studies on the incidence of certain physiological groups of bacteria in the rhizosphere. Can. J. Microbial. 3, 265-269. Loum M. (1966) The effect of microorganisms on the availability of trace elements to plants. Ph.D. thesis, University of Otago.
320
LYNDSAY
FENWICK
LOUTITM. and LoUTIT J. S. (1966) The aerobic heterotrophic bacteria of two New Zealand soils. N.Z. J. agric. Res. 9, 84-92. MARTINJ. P. (1950) Use of acid, rose bengal and streptomycin in the plate method for estimating soil fungi. Soil Sci. 69, 215-232. F?PERC. S. (1950) Soil and Plant Analysis. Waite Agricultural Research Institute, University of Adelaide. ROUATTJ. W., PETERSON E. A., KATZNELSON H. and HENDERSONV. E. (1963) Microorganisms in the root zone in relation to temperature. Can. J. Microbial. 9, 227-236. ROVIRAA. D. (1959) Root excretions in relation to the rhizosphere effect-IV. Influence of plant species, age of plant, light, temperature and calcium nutrition on exudation. PI. Soil 11,53-64. ROVIRA A. D. (1965) Interactions between plant roots and soil microorganisms. A. Rev. Microbial. 19, 241-266. STRZELCZYKE. (1961) Studies on the incidence of certain “nutritional” and physiological groups of bacteria in rhizosphere and non-rhizosphere soil. Acta Microbial. Polon. 10, 169-180. TIMONJN M. I. (1940) The interaction of higher plants and soil microorganisms-I. Microbial population of rhizosphere seedlings of certain cultivated plants. Can. J. Res. C18, 307-317. TIMONINM. I. and THEXTON R. H. (1951) The rhizosphere effect of onion and garlic on the soil microflora. Soil Sci. Sot. Am. Proc. 15,186-189. VIRTANENA. I. and MATIKKALAE. J. (1959) The isolation of S-methylcysteinesulphoxide and S-n-propylcysteinesulphoxide from onion (Allium cepa) and the antibiotic activity of crushed onion. Actu Chem. Stand. 13, 1898-1900.