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Studies on rhizosphere organisms and molybdenum concentration in plants—II. Comparison of isolates from the rhizospheres of plants grown in two soils under the same conditions
Soil Biol. Bicchem. Vol. 4, pp. 267-270. Pergamon Press 1972. Printed in Great Britain
STUDIES ON RHIZOSPHERE ORGANISMS AND MOLYBDENUM CONCENTRATION IN PLANTS-II. COMPARISON OF ISOLATES FROM THE RHIZOSPHERES OF PLANTS GROWN IN TWO SOILS UNDER THE SAME CONDITIONS MARGARET LOUTIT, JENNIFER HILLAS
and G. F. S. SPEARS
Medical School, University of Otago, Dunedin, New Zealand (Accepted 13 March 1972) Summary-The proportions of various genera found in the rhizosphere of the same plant species grown under similar conditions in two soils have been compared. The type of soil and the addition of molybdenum both had an effect. Addition of MO caused an increase in Gramnegative organisms while Gram-positive organisms decreased. INTRODUCTION
grown under identical conditions in Napier and Hastings soil showed significant differences in MO concentration and rhizosphere bacteria in particular, contributed to these differences. As numbers of bacteria were similar in the rhizospheres it was considered necessary to determine the composition of the populations. Methods were devised for handling large numbers of isolates (Bowie, Loutit and Loutit, 1969) and for using computer techniques to facilitate their identification (Loutit, Hillas and Spears, 1973). The isolates were obtained from rhizospheres of plants grown in Napier and Hastings soils, with and without added MO (Loutit et al., 1972). RADISHES
MATERIALS
AND METHODS
The method of growing the experimental plant (Ruphanus sativus L. var. ‘White Icicle’), the amount of MO added, the harvesting of the tops and method of MO determination have been described (Loutit, Malthus and Loutit, 1968; Loutit and Brooks, 1970; Loutit et al., 1972). The procedure for obtaining rhizosphere isolates has also been described as have the steps leading to identification of the isolates (Loutit et al., 1968; Bowie et al., 1969; Loutit et al., 1972). RESULTS
Rhizosphere isolates (1553) were identified to generic level. The results were analyzed to find the percentages of isolates belonging to certain genera present in the rhizospheres of plants grown in the two soils, Napier and Hastings, with and without MO added (Table 1). It was apparent that there were differences in the proportion of certain genera and chisquared tests of association were therefore performed. A number of the differences tested were found to be significant (Tables 2 and 3). Comparing rhizosphere isolates from plants grown in Napier and Hastings soils without any addition of MO it was apparent that numbers of Pseudomonas and Achromobacter were higher in the rhizospheres of plants grown in Hastings soil at the level of significance of 267
268
MARGARET
TABLE
1. NUMBERS
AND
LOUTIT, JENNIFER
PERCENTAGE
HILLAS AND G. F. S. SPEARS
OF ISOLATES BELONGING
PLANTS GROWN
IN
TO DIFFERENT GENERA
Napier No MO added (No. of isolates) (“%)
Genera* Pseudomonas Xanthomonas Protaminobacter Alginomonas Chromobacterium Achromobacter Flavobacterium Erwinia Klebsiella Brevibacterium Corynebacterium Arthrobacter Bacillus Mycobacterium Nocardia Streptomyces Ai B C D E Total
21 16 1 1 0 39 30 1 2 0 10 86 29 0 27 66 11 11 13 11 0
5.60 4.27 0.27 0.27 0.00 10.40 8.00 0.27 0.53 0.00 2.67 22.93 7.73 0.00 7.20 17.60 2.93 2.93 3.47 2.93 0.00
375 -
779-
FROM RHI~~~PHERES 0~
NAPIER AND HASTINGSSOILS Hastings
MO added (No. of isolates) (%) 67 23 0 0
16.58 5.69 0.00 0.00 0.00 23.27 8.42 0.50 0.00 0.00 3.22 12.38 3.71 0.00 0.25 2.23 3,71 4.70 4.46 8.91 1.98
9: 34 2 0 0 13 50 15 0 1 9 15 19 18 36 8 404
No MO added (No. of isolates) (%) 44
10 2 0 0 66 28 5 3 1 6 22 45 1 2 28 8 18 30 10 8 337 -774
MO added (No. of isolates) (%)
13.06 2.97 0.59 0.00 0.00 19.58 8.31 1.48 0.89 0.30 1.78 6.53 13.35 0.30 0.59 8.31 2.37 5.34 8.90 2.97 2.37
52 29 1 0 4 118 29 5 0 3 13 47 25 0 5 25 9 18 33 16 5
11.90 6.64 0.23 0.00 0.92 27.00 6.64 1.14 0.00 0.69 2.97 10.76 5.72 0.00 1.14 5.72 2.06 4.12 7.55 3.66 1.14
437 -
* Genera keyed for and not detected were Mycoplana, Alginobacter, Enterobacter, Hafnia, Proteus, Waksmania, Cellulomonas. t A includes Nocardia, Corynebacterium, Arthrobacter, B includes Corynebacterium, Brevibacterium, Arthrobacter, C includes Enterobacter, Hafnia, Achromobacter, Erwinia, Proteus, D includes FIavobacterium Erwinia, E includes Achromobacter, Erwinia.
TABLE2. CHI-SQUARETESXSTODETERMINE IF THEREWERESIGNIFICANT DIFFERENCES IN DISTRIBUTION OF THOSE GENERA SHOWN IN TABLE1 Napier compared with Hastings
Napier
Hastings
No MO added
No MO added compared with MO added
No MO added compared with MO added
Chi-squared 112.05 Degrees of freedom 13 Level of significance P < 0.001
147.77 13 P < 0.001
32.76 13 P < 0.001
COMPARISON
269
OF RHIZOSPHERE BACTERIA
3. RESULTSOF PHI-~UARE TESTSCOMPARES GENERA FOUNDIN RH~~PHERES OF PLANTSGROWN IN TWO SOILSwrr~ AND WITHOUTADDED MO. TESTSOF RELATIVE FREQUENCYOF INDIVIDUAL GENERA
TABLE
Pseudomonas Xanthomonas Achromobacter Flovobacterium Coryneba~teri~m Arthrobacter Bacillus Nocardia Streptomyces Nocardia Corynebacterium Arthrobacter Corynebacterium Brevibacterium Arthrobacter Enterobacter Achromobacter Er winia Proteus Flavoba~teri~m Erwinia Achromobacter Erwinia *p
Napier compared with Hastings
Napier
Hastings
No MO added (6
No MO compared with MO added (x2)
No MO added compared with MO added
22.33$ o-55
35.85:: 5.43* 18.172 12.57:
0.00 0.05 14.312 5-16* 25.15: 51.06;
0.14 4.6f* s-37* 0.55 0.69 3-68 12.56: 0.17 1.61
0.05
0.16
0.00
2.05
1.20
0.39
s.30t
O-27
o-30
0.38
11-223
o-10
0.80
0.98
11-01: 0.52 11.19:: 0.00 0.29
21.845.
9*77f
W
i= O-05 x2 = 3.841, TP = 0.01 x2 = 6.635, $ P = 0.001 x2 = 10.827.
P = 0~001. Numbers of isolates in the Gram-negative group (Enterobacter, Hafniu, Achromobacter, Erwinia and Proteus) and the Achromobacter-Erwinia group were significantly higher at the level of P = 0.01 and numbers of B~ciZlusisolates were higher at the level of P = 0.05. On the other hand the numbers of isolates within certain other genera were signi-
ficantly higher in the rhizospheres of plants grown in Napier soil. Numbers of isolates of Arthrobacter, No~rd~a and ~treptomyc~s were higher at a significant level of P = 0401. The addition of MO produced some interesting changes in the rhizosphere populations. Addition of MO to Napier soil was associated (P = 0.001) with increases in numbers of Pseudomonas, Achromobacter and the Flavobacterium-Erwinia group, and with decreases in numbers of Arthrobacter, Nocardia and Streptomyces and of Bacillus (P = 0.05). For Hastings grown plants there were also changes following addition of MO. Numbers of Achromobatter and Xanthomonas were higher at the P = O-05 level of significance and numbers of Bacillus lower at the level of P = 0.001. DISCUSSION
We have been able to compare isolates from the rhizospheres of the same plant species grown in two soils of similar origin, but differing in certain characteristics such as pH (Healy, Ludwig and Losee, 1961). It is apparent that the proportion of certain generavaries according to soil type and with the addition of MO. In the rhizospheres of plants grown in
270
MARGARET
LOUTIT, JENNIFER
HILLAS AND G. F. S. SPEARS
Hastings soils the numbers of Gram-negative isolates appeared to be significantly higher than those grown in Napier soil where Gram-positive isolates predominated. Theaddition of MO appeared to resuh in an increase in numbers of Gram-negative isolates from both soils. At the same time the numbers of Gram-positive organisms decreased. Which is the more significant of these changes has yet to be assessed in terms of effect of entry of MO into the plants. The level of significance of the differences is such that in future experiments for comparison of rhizospheres, the numbers of isolates can be considerably reduced. The reported differences in rhizosphere populations of the same plant species grown in different soils under the same conditions may be of considerable importance in a number of fields of research. Acknowfedgemenfs-We are indebted to the staff of the Department of Agriculture, Hastings for cohecting soils and to Tasman Vaccine Laboratory Ltd. for irradiation of soils. The work was supported in part by a
research contract from the Department Palmerston North.
of Scientific and Industrial Research, Applied Biochemistry Division,
REFERENCES BOWIE1. S., LOUTXTM. W. and LOUTITJ. S. (1969) Identi~cation of generic level by using multipoint inocuIation techniques. Can. 1. HEALYW. B., LUDWIGT. G. and LOSEEF. L. (1961) Soils and denta Soif Sci. 92, 359-366. LOUTITM. W. and BRINKS R. R. (1970) Rhizosphere organisms and Soil Biol. Biochem.
aerobic heterotrophic soil bacteria to ~~cru~ioZ. 15, 297-301. caries in Hawke’s Bay New Zealand. molybdenum
concentration
in plants.
2, 131-135.
LOUTITM. W., HILLASJ. L. and SPEARSG. F. S. (1972) Further studies on rhizosphere organisms and molybdenum concentration in plants-I. Identification of rhizosphere isolates to generic level. Soil Biol. B&hem. 4,261-265. LOUTITM. W., MALTHUSR. S. and LOUTITJ. S. (1968) The effect of soil microorganisms on the concentration of molybd~um in the radish ~Ra~h~~us sativus L.) variety ‘White Icicle’. N.Z. J. agric. Res. 11,420-434.
STUDIES ON RHIZOSPHERE ORGANISMS AND MOLYBDENUM CONCENTRATION IN PLANTS-II. COMPARISON OF ISOLATES FROM THE RHIZOSPHERES OF PLANTS GROWN IN TWO SOILS UNDER THE SAME CONDITIONS MARGARET LOUTIT, JENNIFER HILLAS
and G. F. S. SPEARS
Medical School, University of Otago, Dunedin, New Zealand (Accepted 13 March 1972) Summary-The proportions of various genera found in the rhizosphere of the same plant species grown under similar conditions in two soils have been compared. The type of soil and the addition of molybdenum both had an effect. Addition of MO caused an increase in Gramnegative organisms while Gram-positive organisms decreased. INTRODUCTION
grown under identical conditions in Napier and Hastings soil showed significant differences in MO concentration and rhizosphere bacteria in particular, contributed to these differences. As numbers of bacteria were similar in the rhizospheres it was considered necessary to determine the composition of the populations. Methods were devised for handling large numbers of isolates (Bowie, Loutit and Loutit, 1969) and for using computer techniques to facilitate their identification (Loutit, Hillas and Spears, 1973). The isolates were obtained from rhizospheres of plants grown in Napier and Hastings soils, with and without added MO (Loutit et al., 1972). RADISHES
MATERIALS
AND METHODS
The method of growing the experimental plant (Ruphanus sativus L. var. ‘White Icicle’), the amount of MO added, the harvesting of the tops and method of MO determination have been described (Loutit, Malthus and Loutit, 1968; Loutit and Brooks, 1970; Loutit et al., 1972). The procedure for obtaining rhizosphere isolates has also been described as have the steps leading to identification of the isolates (Loutit et al., 1968; Bowie et al., 1969; Loutit et al., 1972). RESULTS
Rhizosphere isolates (1553) were identified to generic level. The results were analyzed to find the percentages of isolates belonging to certain genera present in the rhizospheres of plants grown in the two soils, Napier and Hastings, with and without MO added (Table 1). It was apparent that there were differences in the proportion of certain genera and chisquared tests of association were therefore performed. A number of the differences tested were found to be significant (Tables 2 and 3). Comparing rhizosphere isolates from plants grown in Napier and Hastings soils without any addition of MO it was apparent that numbers of Pseudomonas and Achromobacter were higher in the rhizospheres of plants grown in Hastings soil at the level of significance of 267
268
MARGARET
TABLE
1. NUMBERS
AND
LOUTIT, JENNIFER
PERCENTAGE
HILLAS AND G. F. S. SPEARS
OF ISOLATES BELONGING
PLANTS GROWN
IN
TO DIFFERENT GENERA
Napier No MO added (No. of isolates) (“%)
Genera* Pseudomonas Xanthomonas Protaminobacter Alginomonas Chromobacterium Achromobacter Flavobacterium Erwinia Klebsiella Brevibacterium Corynebacterium Arthrobacter Bacillus Mycobacterium Nocardia Streptomyces Ai B C D E Total
21 16 1 1 0 39 30 1 2 0 10 86 29 0 27 66 11 11 13 11 0
5.60 4.27 0.27 0.27 0.00 10.40 8.00 0.27 0.53 0.00 2.67 22.93 7.73 0.00 7.20 17.60 2.93 2.93 3.47 2.93 0.00
375 -
779-
FROM RHI~~~PHERES 0~
NAPIER AND HASTINGSSOILS Hastings
MO added (No. of isolates) (%) 67 23 0 0
16.58 5.69 0.00 0.00 0.00 23.27 8.42 0.50 0.00 0.00 3.22 12.38 3.71 0.00 0.25 2.23 3,71 4.70 4.46 8.91 1.98
9: 34 2 0 0 13 50 15 0 1 9 15 19 18 36 8 404
No MO added (No. of isolates) (%) 44
10 2 0 0 66 28 5 3 1 6 22 45 1 2 28 8 18 30 10 8 337 -774
MO added (No. of isolates) (%)
13.06 2.97 0.59 0.00 0.00 19.58 8.31 1.48 0.89 0.30 1.78 6.53 13.35 0.30 0.59 8.31 2.37 5.34 8.90 2.97 2.37
52 29 1 0 4 118 29 5 0 3 13 47 25 0 5 25 9 18 33 16 5
11.90 6.64 0.23 0.00 0.92 27.00 6.64 1.14 0.00 0.69 2.97 10.76 5.72 0.00 1.14 5.72 2.06 4.12 7.55 3.66 1.14
437 -
* Genera keyed for and not detected were Mycoplana, Alginobacter, Enterobacter, Hafnia, Proteus, Waksmania, Cellulomonas. t A includes Nocardia, Corynebacterium, Arthrobacter, B includes Corynebacterium, Brevibacterium, Arthrobacter, C includes Enterobacter, Hafnia, Achromobacter, Erwinia, Proteus, D includes FIavobacterium Erwinia, E includes Achromobacter, Erwinia.
TABLE2. CHI-SQUARETESXSTODETERMINE IF THEREWERESIGNIFICANT DIFFERENCES IN DISTRIBUTION OF THOSE GENERA SHOWN IN TABLE1 Napier compared with Hastings
Napier
Hastings
No MO added
No MO added compared with MO added
No MO added compared with MO added
Chi-squared 112.05 Degrees of freedom 13 Level of significance P < 0.001
147.77 13 P < 0.001
32.76 13 P < 0.001
COMPARISON
269
OF RHIZOSPHERE BACTERIA
3. RESULTSOF PHI-~UARE TESTSCOMPARES GENERA FOUNDIN RH~~PHERES OF PLANTSGROWN IN TWO SOILSwrr~ AND WITHOUTADDED MO. TESTSOF RELATIVE FREQUENCYOF INDIVIDUAL GENERA
TABLE
Pseudomonas Xanthomonas Achromobacter Flovobacterium Coryneba~teri~m Arthrobacter Bacillus Nocardia Streptomyces Nocardia Corynebacterium Arthrobacter Corynebacterium Brevibacterium Arthrobacter Enterobacter Achromobacter Er winia Proteus Flavoba~teri~m Erwinia Achromobacter Erwinia *p
Napier compared with Hastings
Napier
Hastings
No MO added (6
No MO compared with MO added (x2)
No MO added compared with MO added
22.33$ o-55
35.85:: 5.43* 18.172 12.57:
0.00 0.05 14.312 5-16* 25.15: 51.06;
0.14 4.6f* s-37* 0.55 0.69 3-68 12.56: 0.17 1.61
0.05
0.16
0.00
2.05
1.20
0.39
s.30t
O-27
o-30
0.38
11-223
o-10
0.80
0.98
11-01: 0.52 11.19:: 0.00 0.29
21.845.
9*77f
W
i= O-05 x2 = 3.841, TP = 0.01 x2 = 6.635, $ P = 0.001 x2 = 10.827.
P = 0~001. Numbers of isolates in the Gram-negative group (Enterobacter, Hafniu, Achromobacter, Erwinia and Proteus) and the Achromobacter-Erwinia group were significantly higher at the level of P = 0.01 and numbers of B~ciZlusisolates were higher at the level of P = 0.05. On the other hand the numbers of isolates within certain other genera were signi-
ficantly higher in the rhizospheres of plants grown in Napier soil. Numbers of isolates of Arthrobacter, No~rd~a and ~treptomyc~s were higher at a significant level of P = 0401. The addition of MO produced some interesting changes in the rhizosphere populations. Addition of MO to Napier soil was associated (P = 0.001) with increases in numbers of Pseudomonas, Achromobacter and the Flavobacterium-Erwinia group, and with decreases in numbers of Arthrobacter, Nocardia and Streptomyces and of Bacillus (P = 0.05). For Hastings grown plants there were also changes following addition of MO. Numbers of Achromobatter and Xanthomonas were higher at the P = O-05 level of significance and numbers of Bacillus lower at the level of P = 0.001. DISCUSSION
We have been able to compare isolates from the rhizospheres of the same plant species grown in two soils of similar origin, but differing in certain characteristics such as pH (Healy, Ludwig and Losee, 1961). It is apparent that the proportion of certain generavaries according to soil type and with the addition of MO. In the rhizospheres of plants grown in
270
MARGARET
LOUTIT, JENNIFER
HILLAS AND G. F. S. SPEARS
Hastings soils the numbers of Gram-negative isolates appeared to be significantly higher than those grown in Napier soil where Gram-positive isolates predominated. Theaddition of MO appeared to resuh in an increase in numbers of Gram-negative isolates from both soils. At the same time the numbers of Gram-positive organisms decreased. Which is the more significant of these changes has yet to be assessed in terms of effect of entry of MO into the plants. The level of significance of the differences is such that in future experiments for comparison of rhizospheres, the numbers of isolates can be considerably reduced. The reported differences in rhizosphere populations of the same plant species grown in different soils under the same conditions may be of considerable importance in a number of fields of research. Acknowfedgemenfs-We are indebted to the staff of the Department of Agriculture, Hastings for cohecting soils and to Tasman Vaccine Laboratory Ltd. for irradiation of soils. The work was supported in part by a
research contract from the Department Palmerston North.
of Scientific and Industrial Research, Applied Biochemistry Division,
REFERENCES BOWIE1. S., LOUTXTM. W. and LOUTITJ. S. (1969) Identi~cation of generic level by using multipoint inocuIation techniques. Can. 1. HEALYW. B., LUDWIGT. G. and LOSEEF. L. (1961) Soils and denta Soif Sci. 92, 359-366. LOUTITM. W. and BRINKS R. R. (1970) Rhizosphere organisms and Soil Biol. Biochem.
aerobic heterotrophic soil bacteria to ~~cru~ioZ. 15, 297-301. caries in Hawke’s Bay New Zealand. molybdenum
concentration
in plants.
2, 131-135.
LOUTITM. W., HILLASJ. L. and SPEARSG. F. S. (1972) Further studies on rhizosphere organisms and molybdenum concentration in plants-I. Identification of rhizosphere isolates to generic level. Soil Biol. B&hem. 4,261-265. LOUTITM. W., MALTHUSR. S. and LOUTITJ. S. (1968) The effect of soil microorganisms on the concentration of molybd~um in the radish ~Ra~h~~us sativus L.) variety ‘White Icicle’. N.Z. J. agric. Res. 11,420-434.