- Email: [email protected]
Abundance of peanut Rhizobium as affected by environmental conditions in Iraq
Soil Bid. Biochem. Vol. 19. No. 4, pp. 391-396. Printed in Great Britain
1987
0038-0717
8’ 53.00 + 0.00
Pergamon Journals Ltd
ABUNDANCE OF PEANUT RHIZOBIUM AS AFFECTED BY ENVIRONMENTAL CONDITIONS IN IRAQ A. N.YOUSEF, A. S. AL-NASSIRI,S. K. AL-AZAWI and N. ABDUL-HUSSAIP*' Agriculture and Water Resources Research Center, Scientific Research Council, P.O. Box 2416. Baghdad, Iraq (Accepted
5 December
1986)
Summary-The effects of several environmental factors on the abundance of peanut rhizobia (Rhkobium spp) were studied in 66 soil samples collected from different locations in Iraq from September 1979 to July 1980. More than 72% of the samples contained this strain of Rhkobium. Samples free of peanut rhizobia were obtained from southern and central regions during winter and summer. Both occurrence and numbers of Rhizobium spp were more pronounced under legumes. Soil conditions suitable for the survival of Rhixbium spp were: organic C, I% or less; electrical conductivity, 2 mmhos cm-’ or less. pH, 7.64. I; lime, 2&30% and cation-exchange capacity, 20-30 m-equiv 100 g-’ soil.
INTRODUCTION
Knowledge of the effects of environmental factors on the population density and activity of the various strains of rhizobia are an essential step in an understanding of the symbiotic nitrogen fixation process. Such factors have been discussed by Lie (1974) and Gibson (1976). We have investigated the influence of organic C content, electrical conductivity (EC), pH, lime content, cation-exchange capacity (CEC), season and plant cover on the abundance of Rhizobium juponicum and Rhizobium spp nodulating peanut plants in Iraqi soils. MATERIALS
AND METHODS
Sampling
A graduated auger was used to collect 66 soil samples from the upper 40 cm layer, from September 1979 to July 1980. The samples represent northern, central and southern regions of Iraq (Fig. I). Some of the meteorological elements and geographical data of these regions are shown in Table I. From each site, at least four samples were collected, thoroughly mixed and a representative sample of 2-3 kg was kept refrigerated until analysis. A IO g lot was used for counting rhizobia while the remainder was air dried, pulverized and used for the chemical determinations. Rhizobial count
Undamaged and, as uniform as possible, seeds of peanut, Arachus hypogaea cv. Giza Madad, and soybean, Glycine max cv. Lee, were first surface sterilized with 0.2% HgClz and 95% ethanol (Vincent, 1970) and then set to germinate in Petri-dishes containing nutrient agar. Three seedlings were then planted in each of autoclaved Leonard-jar assemblies (Vincent, 1970) and thereafter thinned to one seedling per assembly.
From each soil sample, serial dilutions, ranging from 10-r to 10e6 were prepared and added at a rate of I ml per assembly. After 6 weeks at 26 i_ 4’C in a greenhouse the numbers of nodulated plants were recorded and the most probable number (MPN) of Rhizobium was estimated for each soil sampled (Brockwell, 1963). Chemical analysis
Saturated soil extracts were used for measuring the electrical conductivity (EC) and soil reaction (pH). For the cation-exchange capacity (CEC) and lime content determinations, the methods described by USDA Salinity Laboratory Staff (19.54) were used. Organic C content was determined using the Walkley and Black’s method (1934). All analyses were performed in triplicate. RESULTS
Abundunce of R. japonicum
The bacteriological examinations none of the 66 soil samples contained Abundunce of Rhizobium
showed
that
R. juponicum.
spp (peanut)
Data, shown in Table 2 reveal that only 48 samples (more than 72%) contained peanut rhizobia. The number varied; IO samples had < 100 cells g-t soil, while I8 samples contained IO-IOOOg-‘. However, 30% of the samples had a density of > 1000 cells g-r. These findings indicate that Iraqi soils are fairly well populated with peanut rhizobia nodulating Giza Madad plants, the only cultivar examined in the study. Effect of organic carbon content
Most of the studied samples (58 samples) contained organic carbon ranging from < 0.5 to 1% (Table 3). Only 26% of these samples were devoid of Rhizobium. At a level higher than I % organic carbon (8 samples), five were inhabited by Rhizobium. 391
392
A. N.
YCXSEF
-\
er ul.
.~AG~DAD
\
I
Bas 1’
0~5~CKl
Km
Fig. I. Location and number of soil sampies obtained from difftxent regions of Iraq. North: Al-Tameem and Nineveh: Central: Diyaki, Baghdad, Babel, Wasit: Southern: Basrah, Missan and Kadesiah.
Table I. Mean monthly meter&&d Jai-l. Mosul Baghdad Basrah
Feb.
March
and geo~r~phi~31 data (30 yr records) of the three Iraqi regions (~d~p[ed from frqi Organization. 1979)” Amil
12.4
8.7 12.3 14.6
12.3 16.3 lI1.7
17.4 21.9 24. I
MOSUl Baghdad Basra h
82 71 7s
76 62 70
70 52 64
6’ 44 58
MOSUI
67.3 25.4 22.5
64.2 24.2 13.8
69.6 23.7 20.2
50.g 22.3 20.4
Baghdad Barrah
Mosul Baghdad Basrah
7.0
IO.0
Latitude 36 I9 33 I4 30 34
MlV
JUIK
JUlV
Temperarure ( C)D 24. I 30.5 34.0 28.9 33.0 34.0 29.7 32.7 34.0 Relative humidity (%)” 4-l 30 26 31 2’ 23 53 49 49 Precipitation fmmjh 24.7 0.7 0. I g.1 0.1 tr 7.8 tr 0. I Geographical data Longitude 43 90 44 I4 42 47
Aug.
Sept.
Oct.
Nov.
Dec.
33.0 34.4 33.6
27.7 30.6 30.6
20 5 24.6 25.9
13.5 17.1 19.3
8.3 Il.0 13.6
29 ‘1 48
34 27 SO
49 36 56
67 56 69
82 71 78
36.1 17.2 22.R
67.3 22.9 30.3
lr tr
0.7 0.3
tr
1r
9.9 3.7 I.0
Meteorciogic;ll
Annual (mm) 391.9 147.9 138.9
Elevation (m) 223 34 2
‘Data obtained from Mosul. Baghdad and Basrrth mereroiogicnl st;rtions a-hich reprerenc the norrhern. central and southern regions. respectivef~. from where the soil samples were coilectrd. “Mran average of 30yr.
393
Peanut rhizobia in Iraqi soils Table
2. Number
of
Rhirobium
peanut
sampledfrom September1979
soils
occurrence of the Rhkobium spp studied was 7.68. I since 87% (41 samples) of the rhizobia-inhabited samples were within this range. Most of these samples (25) contained < 1000 ceils g-’ (Table 5).
in
to July
1980 Counts
g-’
soil
No.
of samples
0
18 (27)l
I-IO
3(5)
I&l00
7(l1) 18 (27)
100-IO00 >lwO No.
of
Effect of lime content
Among the 53 samples, with a lime content ranging from >20 to 40%, only 13 were free of Rhizobium spp (Table 6). The bacterial density of most of the inhabited samples (26) was < IOOOg-‘. The lime content of 54% of these samples was >20-30%. Also, most of the samples with a population density of > 1000 g-’ were found to have a similar lime concentration range.
20 (30) 66
samples
‘Percentage
of samples
shown
in parenthe-
sis.
Effect of electrical conductivity
As clearly shown in Table 4, one-third of the samples free of peanut rhizobia had an EC of >4mmhoscm‘. The EC of most of the populated samples (87%) was found to range from
EJect of CEC
All the 43 soil samples, harboured peanut rhizobia, were located in the CEC range of > IO-30 m-equiv 100 g-’ soil (Table 7). It is evident that > 10-20 m-equiv 100 g-’ was the most suitable CEC range for the survival of the peanut Rhizobium. The number of samples, devoid of the bacteria, represents only I I % of the total number having such CEC range which, in general, represents the lowest number compared with the other CEC values.
Eflecr of pH
It seems that the most suitable pH range for the
Table
of Rhtohium
3. Abundance
spp (panul)
as atkted
from
September
No.
of samples
Counts g _’ soil
No.
amounls
8 (20)
I (33) 2 (67)
>I000
8 (40) 20
l5W) I5 (40) 38
shown
in parenthesis.
of samples
4. Abundance
Rhi:ohium
of
‘)
Counls
No.
soil
0 < 1000 No.
0
2(67P
O(0)
3(IOO)
lOcO
O(0) I (33)
O(0) 0 (0)
O(0) O(0)
No.
3
0
3
of samples
shown
25 (38)
5
66
as afiected
by the electrical
Scptembcr
1979
different
(EC)
lo July
conductivity
1980 No.
levels >4
of
samoles
3 (60)
3 (75)
6 (67)
18 (27)
6 (37)
2 (40)
I (25)
I (II)
26 (40)
6 (37)
0 (0)
0 (0)
2 (22)
22 (33)
5
4
9
66
lb
shown
No. 7.3
2 (40)
4 (26)
spp (peanut)
7.2
23 (35)
2 (6) 14(41)
7.1
18 (27)
I(Z0)
>3-l
32
of Rhk-obium
5. Abundance
of samples
2 (40)
> 2-3
of samples
Count
from
7.4
of
samples
> l-2
lb(53)
of samples
> I .s-2.0%
> lo00
‘Percentage
spp (peanut)
in soils sampled
of
0 (0) 3
in soils sampled
(%)
No.
7 (35)
of snmoles
content
> l.O-1.5%
5 (25)
a-’
of
carbon
0
(mmhoscm
soil
differen
carbon
1980
cl000
Table
g-’
with
organic >os-1.0%
<0.5%
*Percentage
Table
by organic
1979 IO July
in parenlhesis
as affected
of samples 7.5
by the soil pH
with
different
pH
in soils sampled
from
September
1979 IO July
values
No.
7.6
7.7
7.8
7.9
8.0
8.1
8.2
8.3
8.4
Table
3(60)
2(20)
O(0)
4(27)
O(0)
2(29)
I(l4)
O(0)
I (50) O(O)
18 (27)
I (0)
2(40)
3(30)
2(50)
8(53)
b(86)
2(29)
4(57)
O(0)
0 (0)
3(42)
2(29)
I(lo0)
O(0) I(lO0)
28 (43)
O( 100)
O(0) I(50)
I
5
:(50)
l;(2O)
7
7
I
2
I
66
5(50) IO
;(l4)
in parenthesis.
6. Abundance
of Rhbohium
spp (peanut) September
Counts g
No.
’
No.
soil
of samples
< IO%
> I O-20%
as affected
with
different
> 20-30%
0
2 (67)
2(29)
I2 (32)
I (33)
2 (29)
I4 (36)
z lo00
0 (0)
of samples
3
of samples
3 (42)
12(32)
~’
7‘
’
shown
in parenthesis.
by the lime content
1979 to July
‘Percenlage
of
samples
O(0)
samples ‘Percentage
19X0
38‘
(%)
in soils sampled
from
1980 amounts
of lime
> 3&40%
I
(7) 8 (53) 6 (40) 15‘
No. 5 40%
I
(33) 0 (0)
of
samples I8 (27) 25 (38)
2 (67)
23 (35)
3‘
66‘
20(30)
394
A.
Table 7. Abundance
Rhtobium
of
spp (peanut) from
Counts
No.
No.
Table
0 (0)
IO (32)
0 (0) I(IO0)
>I000
II
of samples
6
27
of
Rhkobium
(41)
in sods sampled
levels of CEC
I2 (39)
l(17) 2 (33)
8. Abundance
different
3(11) 13 (45)
3 (SO)
shown
with
100 g-‘)
1980
z D-30
0
of samples
1979 to July
> IO-20
‘Percentage
as affected by the CEC (m-equiv
September
of samples
g-’
YOCSEFer al
N.
No.
>3a&l
9 (29)
>40
I
31
of
samples
O(0)
IS (27)
I (loo)
28 (38)
0 (0) I
20 (35) 66
in parenthesis.
spp (peanut)
as affected
by the date of sampling
in soild sampled
from
September
1979 to July
1980 Coun1s I-’
No.
soil
SetYt.
No.
Mav
JWle
Julv
0 (0)
0 (0)
7 (58)
3 (75)
I8 (27)
4 (75)
3 (75)
3 (25)
I (25)
28 (43)
2(17)
0 (0)
20 (30)
4
66
cl(0)
0 (0)
7 (39)
I(l7)
4 (36)
3 (75)
7 (39)
3 (50)
4 (22)
2 (33)
I (25)
1(25)
6
5
4
2(100)
7 (61)
1(25)
2
II
4
shown
I8
of
months
0 (0)
Jan.
No.
different Feb.
0 (0)
of samples of samples
during
Dec.
>lOQO
‘Percentage
collected
Nov.
0 < 1000
of samples
ocl.
I?
samoles
in parenthesis.
Eflect of data of sampling
Eflect of sampling location
All samples, lacking Rhi:obium spp, were taken either in December and January or in June and July (Table 8). More than 66% of the December-January samples were populated with peanut rhizobia, while 37% of the June-July samples lacked rhizobia.
All the soil samples, devoid of Rhkobium spp were collected either from southern (14 out of 17) or central (4 out of 43) regions (Table IO). None of the southern samples had more than 1000 cells g-‘. On the contrary, more than 33% of the northern region samples were populated with > 1000 cellsg-‘. In the central region, the number of samples with a density of < 1000 or > 1000 rhizobia g-’ was, more or Icss. the same.
Effect of cropping system Table 9 shows that peanut rhizobia were present in fallow and cultivated soils. On the other hand, the presence of rhizobia varied in accordance with the type of crop. The percentage of rhizobia-free samples was 13 with legumes, 33 with non-leguminous crops, vegetables and fallow, and 22 with orchard trees. In addition, rhizobia-containing samples, collected from soils bearing legumes, IO out of 13 (more than 76%) had > 1000 cells g-’ which was the highest amongst all types of cropping systems. Unfortunately, the cropping histories of most of the sampling sites were not available. Such data would have provided information on the survival and population build-up of rhizobia in time.
Table
9. Abundance
Rhizobium
of
spp (peanut)
from No.
Counts
g ’
No.
soil
Legume
The absence of R. japonicrtm from all of the 66 soil samples studied could be explained by the fact that the host plant genus, G/ycine, is not common in Iraq. Glycine max has only recently been introduced here and is only grown on experimental scale. Moreover, only one soybean cultivar (Lee) has been tested in this work. Examination of other crop cultivars is essential to confirm these results.
as affected
September
of samples
DISCUSSION
under
Non-legume
by the cropping
1979 to July different
systrm
in soils sampled
1980 cropping
NO.
system
Orchards
Vegetables
Fallow
oi
samples
0
2(13)
7 (33
2 (22)
5 (33)
2 (33)
I8 (27)
3 (20)
IO (48)
4 (45)
8 (54)
3 (SO)
2x (43)
4(19) 21
3 (33) 9
I(17) 6
66
>I000
lO(67)
of samples
I5
‘Percentage
of samples
Table
shown
IO. Abundance
of
Rhizobium
from
No.
Northern
spp (peanut)
from No.
Counts soil
20 (30)
in parenthesis.
in soils sampled
g -’
2(l3) I5
as atTected by the sampling
September
of samples different
1979 to July
location
1980.
collected locations
No.
Central
Southern
of
samples
0
0 (0)’
18(?7)
4 (67)
4 (9) 21(49)
I4 (82)
cl000
3(18)
28 (43)
> 1000
2 (33) 6
I8 (42) 43
0 (0) I7
of samples
‘Percentage
of samples
shown
in parenthesis
20 (30) 66
Peanut rhizobia
In contrast to R. japonicum. Rhj~ob~um spp were found in many of the soil samples. These strains can nodulate many legumes such as peanut. Arachus hFpogaea. cowpea, Vigna Sirtensis lima bean, Phuseolus lunatus and others. Many of these plants are grown widely in Iraq. The absence or low numbers of Rhizobium spp found in the salt-affected soil samples (EC more than 4mmhos cm-‘) is consistent with the findings of Piliai and Sen (1966) on R. trifalii, Yadav and Vyas (1973) on R. melifuri, Steinborn and Roughley (1975) on R. [egttmi~osu~urn (nodulating peas), Ty (1981) on R. juponicttm and (A. N. Yousef, unpublished Ph.D. thesis, Dundee University, 1982) on R. feguminosarum nodulating field beans). The limited presence of Rhi:obitcm spp in the soil samples having more than I % organic carbon (Table 2) could be attributed to an effect, caused by the relatively high organic matter content, which activates other soil microflora such as fungi, actinomycetes and protozoa. One or more of these organisms might have an antagonistic effect on the rhizobial growth. Such effects, in fact have been reported by Damirgi and Johnson (1966). Moreover, during the decomposition of organic matter in the soil, some of the compounds released may either have a toxic effect on the bacteria or cause unfavourablc conditions for their growth and activity. According to Mulder and Van Veen (1960) there are unknown organic compounds that might have lethal effects on Rhixbittm in the soil. The fact that more than 85% of the populated samples had a pH value of 7.6-8. I could be explained by two masons. First, as soil bacteria, rhizobia in general, prefer to grow under neutral or slightly alkaline conditions. The second, is that such a range of pH is the most common one in Iraqi soils which is. therefore, expected to bc the favourable range for the occurrence of the indigenous microorganisms. The CEC of any soil varies with the amount and type of clay and organic matter. In the present study, although soil texture was not dctcrmincd. variations in the CEC could bc mainly attributed to the clay content of the soil samples since the organic carbon content was very low (86% of the whole samples contained <0.5-l % organic carbon, Table 3). Thcrefort, organic matter probably had no effect on the CEC value However. the effect of clay content on enhancing the survival of root-nodule bacteria have been documcntcd by Vyas and Prasad (1960) and Marshall (1968, 1975). Differences in the counts of Rlri-_ohilrnrspp, found under various cropping systems. could be due to various cultivation practices. According to Alexander (l96l), plowing and tillage operations are drastic environmental treatments that usually caused marked ba~tcriologi~l alterations. in addition, secretions of different plant-root systems may affect both type and density of soil microflora. Such effects, have been reported by Rovira and 1McDougall (1967). The evidence shown in Table 8 that the prcsencc of rhizobia was more affected during summer than in winter was cxpcctcd. In Iraq. mainly in the central and southern regions, the daily hig.h temperature along with the decrease in soil motsturc content, during summer months, could produce a partial
in Iraqi soils
395
ste~Iization effect in at least the upper IOcm soil layer. Similar findings have been also shown by Taylor and Burns (1924) and Abed-el-Malek and Rizk (1966). On the other hand low winter temperatures may only check bacterial activities but not destroy their cells. Therefore, the absence of Rhizobium spp from samples collected during December and January, might be a result of either a real absence of the bacteria or to a harmful effect of the preceding summer. To be certain there is any significant interactive effect between the peanut rhizobial number and some of the studied factors, the data were submitted to computer analysis using a multiple planer regression program. The correlation coefficients were: 0. I72 for the effect of data of sampling, geographical location and climatic factors, 0.318 for the effect of lime content and pH and 0.421 for electrical conductivity and cation-exchange capacity. Therefore, the only significant correfation was found between electrical conductivity and cation-exchange capacity (and rhizobial numbers) (N = 66, r = 0.36 and z = 0.1 Oh). The present investigation showed that, in Iraq. inoculation of soybean seeds with R. joponictmt is essential since the Rhizobium was not detected in any of the soil samples studied. Despite the fact that peanut rhizobia (Rlti:obt’trnt spp) were found in most of the samples, their occurrence and population were affected by several environmental factors. Since R~ti~i~bit~~tspp found here are indigenous, studies concerning their efficiency compared with other introduced strains, are of obvious importance for studies of symbiotic N,-fixation. REFERENCES Abed-el-Malek
Y. and Rizk S. G. (1966)
Microbial and soils. Jo~cmcrl of .tfkro1. 47-56. inrro&ction IO Soil Microhidc~~y.
nitrogen changes in Sharaqi bido~.v. ffttired Aruh Repubk
Alexander Wiley.
M.
( i 96 I )
New York.
Brockwcll 1. (1963) Accuracy of plant for counting Microbiology
population of 11, 377-383.
Rhixhm
infection tcchniqucs rr(/b/ii. App/ic*rl
Johnson H. W. (1966) Elkt of soil actinomycets on strains of Ritixhium jupcmirurtl. .J,qrrtt~onty Journnl 58, ‘23-226. Gibson A. H. (1976) Limitation to dinitrogcn fixation by legumes. Proceedings. Firsr In~~rn~~il~n~l S~~i~f).s;~~~~lott Nitrogen Fi.wtiwr (W. E. Newton and C. J. Nyman, Eds). pp. 4004128. Washington State University, Pullman,. Lie T. A. (1974) Environmental effects on nodulation and symbiotic nitrogen fixation. In Talc Bio/o& of ?V;irro,cytr Fi.wrion (A. Quispel. Ed.). pp. 555-582. North Holland. Amsterdam. Marshall K. C. (1968) Interaction between colloidal montmorillonite and cells of Rhizohium spccics w-ith different ionogenic surfaces. ~~~~~~z~#ri~~Bj(/p~f~.si~~iAcrcl 156, 179-168. Marshall K. C. (1975) Clay mineralogy in relation to survival of soil bacteria. Annual Rekw of Plryropurho/o~ty 13. 357-373. Mulder E. G. and Van Veen N. L. (1960) EKect of oH and organic compounds on nitrogen fixation by red’clover. Phnr and Soil 113, 91-l 13. Pillai R. N. and Sen A. (1966) Salt tolerance of R. rr@i. Indian Jmmd of Agriculture Stirnces 36, 80--8-t. Rovira A. D. and McDougall B. M. (1967) Microbiological and biochemical aspects of the rhizosphere. In %ii Damirgi S. M. and
396
A. N. YOCSEF er al.
Biochemistry (A. D. McLaren and G. F. Peterson. Eds). pp. 417-463. Dekker, New York. Steinborn J. and Roughley R. J. (I 975) Toxicity of sodium and chloride ions to Rhizobium spp in broth and peat cultures. Journal of Applied Bacteriology 39, 133-138. Taylor E. M. and Burns A. G. (1924) Soil temperatures during sharaqi period and their agricultural significance. Ministry of Agriculture, Egypt. Technical Bulletin No. 31. Tu J. C. (1981) Effect of salinity on Rhizobium root-hair interaction, nodulation and growth of soybean. Canadian Journal of Plant Sciences 61, 231-239. USDA Salinity Laboratory Staff (1954) Diagnosis and Improvemenr of Saline and Alkali Soils. Handbook No. 60, USDA, Washington.
Van Schreven D. C. (I 964) The effect of some actinomycetes on rhizobia and .4grobacrerium radiobacrer. Plant and Sod 21. 283-302. Vincent J. M. (1970) d Manualfor the Practical Stu
1987
0038-0717
8’ 53.00 + 0.00
Pergamon Journals Ltd
ABUNDANCE OF PEANUT RHIZOBIUM AS AFFECTED BY ENVIRONMENTAL CONDITIONS IN IRAQ A. N.YOUSEF, A. S. AL-NASSIRI,S. K. AL-AZAWI and N. ABDUL-HUSSAIP*' Agriculture and Water Resources Research Center, Scientific Research Council, P.O. Box 2416. Baghdad, Iraq (Accepted
5 December
1986)
Summary-The effects of several environmental factors on the abundance of peanut rhizobia (Rhkobium spp) were studied in 66 soil samples collected from different locations in Iraq from September 1979 to July 1980. More than 72% of the samples contained this strain of Rhkobium. Samples free of peanut rhizobia were obtained from southern and central regions during winter and summer. Both occurrence and numbers of Rhizobium spp were more pronounced under legumes. Soil conditions suitable for the survival of Rhixbium spp were: organic C, I% or less; electrical conductivity, 2 mmhos cm-’ or less. pH, 7.64. I; lime, 2&30% and cation-exchange capacity, 20-30 m-equiv 100 g-’ soil.
INTRODUCTION
Knowledge of the effects of environmental factors on the population density and activity of the various strains of rhizobia are an essential step in an understanding of the symbiotic nitrogen fixation process. Such factors have been discussed by Lie (1974) and Gibson (1976). We have investigated the influence of organic C content, electrical conductivity (EC), pH, lime content, cation-exchange capacity (CEC), season and plant cover on the abundance of Rhizobium juponicum and Rhizobium spp nodulating peanut plants in Iraqi soils. MATERIALS
AND METHODS
Sampling
A graduated auger was used to collect 66 soil samples from the upper 40 cm layer, from September 1979 to July 1980. The samples represent northern, central and southern regions of Iraq (Fig. I). Some of the meteorological elements and geographical data of these regions are shown in Table I. From each site, at least four samples were collected, thoroughly mixed and a representative sample of 2-3 kg was kept refrigerated until analysis. A IO g lot was used for counting rhizobia while the remainder was air dried, pulverized and used for the chemical determinations. Rhizobial count
Undamaged and, as uniform as possible, seeds of peanut, Arachus hypogaea cv. Giza Madad, and soybean, Glycine max cv. Lee, were first surface sterilized with 0.2% HgClz and 95% ethanol (Vincent, 1970) and then set to germinate in Petri-dishes containing nutrient agar. Three seedlings were then planted in each of autoclaved Leonard-jar assemblies (Vincent, 1970) and thereafter thinned to one seedling per assembly.
From each soil sample, serial dilutions, ranging from 10-r to 10e6 were prepared and added at a rate of I ml per assembly. After 6 weeks at 26 i_ 4’C in a greenhouse the numbers of nodulated plants were recorded and the most probable number (MPN) of Rhizobium was estimated for each soil sampled (Brockwell, 1963). Chemical analysis
Saturated soil extracts were used for measuring the electrical conductivity (EC) and soil reaction (pH). For the cation-exchange capacity (CEC) and lime content determinations, the methods described by USDA Salinity Laboratory Staff (19.54) were used. Organic C content was determined using the Walkley and Black’s method (1934). All analyses were performed in triplicate. RESULTS
Abundunce of R. japonicum
The bacteriological examinations none of the 66 soil samples contained Abundunce of Rhizobium
showed
that
R. juponicum.
spp (peanut)
Data, shown in Table 2 reveal that only 48 samples (more than 72%) contained peanut rhizobia. The number varied; IO samples had < 100 cells g-t soil, while I8 samples contained IO-IOOOg-‘. However, 30% of the samples had a density of > 1000 cells g-r. These findings indicate that Iraqi soils are fairly well populated with peanut rhizobia nodulating Giza Madad plants, the only cultivar examined in the study. Effect of organic carbon content
Most of the studied samples (58 samples) contained organic carbon ranging from < 0.5 to 1% (Table 3). Only 26% of these samples were devoid of Rhizobium. At a level higher than I % organic carbon (8 samples), five were inhabited by Rhizobium. 391
392
A. N.
YCXSEF
-\
er ul.
.~AG~DAD
\
I
Bas 1’
0~5~CKl
Km
Fig. I. Location and number of soil sampies obtained from difftxent regions of Iraq. North: Al-Tameem and Nineveh: Central: Diyaki, Baghdad, Babel, Wasit: Southern: Basrah, Missan and Kadesiah.
Table I. Mean monthly meter&&d Jai-l. Mosul Baghdad Basrah
Feb.
March
and geo~r~phi~31 data (30 yr records) of the three Iraqi regions (~d~p[ed from frqi Organization. 1979)” Amil
12.4
8.7 12.3 14.6
12.3 16.3 lI1.7
17.4 21.9 24. I
MOSUl Baghdad Basra h
82 71 7s
76 62 70
70 52 64
6’ 44 58
MOSUI
67.3 25.4 22.5
64.2 24.2 13.8
69.6 23.7 20.2
50.g 22.3 20.4
Baghdad Barrah
Mosul Baghdad Basrah
7.0
IO.0
Latitude 36 I9 33 I4 30 34
MlV
JUIK
JUlV
Temperarure ( C)D 24. I 30.5 34.0 28.9 33.0 34.0 29.7 32.7 34.0 Relative humidity (%)” 4-l 30 26 31 2’ 23 53 49 49 Precipitation fmmjh 24.7 0.7 0. I g.1 0.1 tr 7.8 tr 0. I Geographical data Longitude 43 90 44 I4 42 47
Aug.
Sept.
Oct.
Nov.
Dec.
33.0 34.4 33.6
27.7 30.6 30.6
20 5 24.6 25.9
13.5 17.1 19.3
8.3 Il.0 13.6
29 ‘1 48
34 27 SO
49 36 56
67 56 69
82 71 78
36.1 17.2 22.R
67.3 22.9 30.3
lr tr
0.7 0.3
tr
1r
9.9 3.7 I.0
Meteorciogic;ll
Annual (mm) 391.9 147.9 138.9
Elevation (m) 223 34 2
‘Data obtained from Mosul. Baghdad and Basrrth mereroiogicnl st;rtions a-hich reprerenc the norrhern. central and southern regions. respectivef~. from where the soil samples were coilectrd. “Mran average of 30yr.
393
Peanut rhizobia in Iraqi soils Table
2. Number
of
Rhirobium
peanut
sampledfrom September1979
soils
occurrence of the Rhkobium spp studied was 7.68. I since 87% (41 samples) of the rhizobia-inhabited samples were within this range. Most of these samples (25) contained < 1000 ceils g-’ (Table 5).
in
to July
1980 Counts
g-’
soil
No.
of samples
0
18 (27)l
I-IO
3(5)
I&l00
7(l1) 18 (27)
100-IO00 >lwO No.
of
Effect of lime content
Among the 53 samples, with a lime content ranging from >20 to 40%, only 13 were free of Rhizobium spp (Table 6). The bacterial density of most of the inhabited samples (26) was < IOOOg-‘. The lime content of 54% of these samples was >20-30%. Also, most of the samples with a population density of > 1000 g-’ were found to have a similar lime concentration range.
20 (30) 66
samples
‘Percentage
of samples
shown
in parenthe-
sis.
Effect of electrical conductivity
As clearly shown in Table 4, one-third of the samples free of peanut rhizobia had an EC of >4mmhoscm‘. The EC of most of the populated samples (87%) was found to range from
EJect of CEC
All the 43 soil samples, harboured peanut rhizobia, were located in the CEC range of > IO-30 m-equiv 100 g-’ soil (Table 7). It is evident that > 10-20 m-equiv 100 g-’ was the most suitable CEC range for the survival of the peanut Rhizobium. The number of samples, devoid of the bacteria, represents only I I % of the total number having such CEC range which, in general, represents the lowest number compared with the other CEC values.
Eflecr of pH
It seems that the most suitable pH range for the
Table
of Rhtohium
3. Abundance
spp (panul)
as atkted
from
September
No.
of samples
Counts g _’ soil
No.
amounls
8 (20)
I (33) 2 (67)
>I000
8 (40) 20
l5W) I5 (40) 38
shown
in parenthesis.
of samples
4. Abundance
Rhi:ohium
of
‘)
Counls
No.
soil
0 < 1000 No.
0
2(67P
O(0)
3(IOO)
O(0) I (33)
O(0) 0 (0)
O(0) O(0)
No.
3
0
3
of samples
shown
25 (38)
5
66
as afiected
by the electrical
Scptembcr
1979
different
(EC)
lo July
conductivity
1980 No.
levels >4
of
samoles
3 (60)
3 (75)
6 (67)
18 (27)
6 (37)
2 (40)
I (25)
I (II)
26 (40)
6 (37)
0 (0)
0 (0)
2 (22)
22 (33)
5
4
9
66
lb
shown
No. 7.3
2 (40)
4 (26)
spp (peanut)
7.2
23 (35)
2 (6) 14(41)
7.1
18 (27)
I(Z0)
>3-l
32
of Rhk-obium
5. Abundance
of samples
2 (40)
> 2-3
of samples
Count
from
7.4
of
samples
> l-2
lb(53)
of samples
> I .s-2.0%
> lo00
‘Percentage
spp (peanut)
in soils sampled
of
0 (0) 3
in soils sampled
(%)
No.
7 (35)
of snmoles
content
> l.O-1.5%
5 (25)
a-’
of
carbon
0
(mmhoscm
soil
differen
carbon
1980
cl000
Table
g-’
with
organic >os-1.0%
<0.5%
*Percentage
Table
by organic
1979 IO July
in parenlhesis
as affected
of samples 7.5
by the soil pH
with
different
pH
in soils sampled
from
September
1979 IO July
values
No.
7.6
7.7
7.8
7.9
8.0
8.1
8.2
8.3
8.4
Table
3(60)
2(20)
O(0)
4(27)
O(0)
2(29)
I(l4)
O(0)
I (50) O(O)
18 (27)
I (0)
2(40)
3(30)
2(50)
8(53)
b(86)
2(29)
4(57)
O(0)
0 (0)
3(42)
2(29)
I(lo0)
O(0) I(lO0)
28 (43)
O( 100)
O(0) I(50)
I
5
:(50)
l;(2O)
7
7
I
2
I
66
5(50) IO
;(l4)
in parenthesis.
6. Abundance
of Rhbohium
spp (peanut) September
Counts g
No.
’
No.
soil
of samples
< IO%
> I O-20%
as affected
with
different
> 20-30%
0
2 (67)
2(29)
I2 (32)
I (33)
2 (29)
I4 (36)
z lo00
0 (0)
of samples
3
of samples
3 (42)
12(32)
~’
7‘
’
shown
in parenthesis.
by the lime content
1979 to July
‘Percenlage
of
samples
O(0)
samples ‘Percentage
19X0
38‘
(%)
in soils sampled
from
1980 amounts
of lime
> 3&40%
I
(7) 8 (53) 6 (40) 15‘
No. 5 40%
I
(33) 0 (0)
of
samples I8 (27) 25 (38)
2 (67)
23 (35)
3‘
66‘
20(30)
394
A.
Table 7. Abundance
Rhtobium
of
spp (peanut) from
Counts
No.
No.
Table
0 (0)
IO (32)
0 (0) I(IO0)
>I000
II
of samples
6
27
of
Rhkobium
(41)
in sods sampled
levels of CEC
I2 (39)
l(17) 2 (33)
8. Abundance
different
3(11) 13 (45)
3 (SO)
shown
with
100 g-‘)
1980
z D-30
0
of samples
1979 to July
> IO-20
‘Percentage
as affected by the CEC (m-equiv
September
of samples
g-’
YOCSEFer al
N.
No.
>3a&l
9 (29)
>40
I
31
of
samples
O(0)
IS (27)
I (loo)
28 (38)
0 (0) I
20 (35) 66
in parenthesis.
spp (peanut)
as affected
by the date of sampling
in soild sampled
from
September
1979 to July
1980 Coun1s I-’
No.
soil
SetYt.
No.
Mav
JWle
Julv
0 (0)
0 (0)
7 (58)
3 (75)
I8 (27)
4 (75)
3 (75)
3 (25)
I (25)
28 (43)
2(17)
0 (0)
20 (30)
4
66
cl(0)
0 (0)
7 (39)
I(l7)
4 (36)
3 (75)
7 (39)
3 (50)
4 (22)
2 (33)
I (25)
1(25)
6
5
4
2(100)
7 (61)
1(25)
2
II
4
shown
I8
of
months
0 (0)
Jan.
No.
different Feb.
0 (0)
of samples of samples
during
Dec.
>lOQO
‘Percentage
collected
Nov.
0 < 1000
of samples
ocl.
I?
samoles
in parenthesis.
Eflect of data of sampling
Eflect of sampling location
All samples, lacking Rhi:obium spp, were taken either in December and January or in June and July (Table 8). More than 66% of the December-January samples were populated with peanut rhizobia, while 37% of the June-July samples lacked rhizobia.
All the soil samples, devoid of Rhkobium spp were collected either from southern (14 out of 17) or central (4 out of 43) regions (Table IO). None of the southern samples had more than 1000 cells g-‘. On the contrary, more than 33% of the northern region samples were populated with > 1000 cellsg-‘. In the central region, the number of samples with a density of < 1000 or > 1000 rhizobia g-’ was, more or Icss. the same.
Effect of cropping system Table 9 shows that peanut rhizobia were present in fallow and cultivated soils. On the other hand, the presence of rhizobia varied in accordance with the type of crop. The percentage of rhizobia-free samples was 13 with legumes, 33 with non-leguminous crops, vegetables and fallow, and 22 with orchard trees. In addition, rhizobia-containing samples, collected from soils bearing legumes, IO out of 13 (more than 76%) had > 1000 cells g-’ which was the highest amongst all types of cropping systems. Unfortunately, the cropping histories of most of the sampling sites were not available. Such data would have provided information on the survival and population build-up of rhizobia in time.
Table
9. Abundance
Rhizobium
of
spp (peanut)
from No.
Counts
g ’
No.
soil
Legume
The absence of R. japonicrtm from all of the 66 soil samples studied could be explained by the fact that the host plant genus, G/ycine, is not common in Iraq. Glycine max has only recently been introduced here and is only grown on experimental scale. Moreover, only one soybean cultivar (Lee) has been tested in this work. Examination of other crop cultivars is essential to confirm these results.
as affected
September
of samples
DISCUSSION
under
Non-legume
by the cropping
1979 to July different
systrm
in soils sampled
1980 cropping
NO.
system
Orchards
Vegetables
Fallow
oi
samples
0
2(13)
7 (33
2 (22)
5 (33)
2 (33)
I8 (27)
3 (20)
IO (48)
4 (45)
8 (54)
3 (SO)
2x (43)
4(19) 21
3 (33) 9
I(17) 6
66
>I000
lO(67)
of samples
I5
‘Percentage
of samples
Table
shown
IO. Abundance
of
Rhizobium
from
No.
Northern
spp (peanut)
from No.
Counts soil
20 (30)
in parenthesis.
in soils sampled
g -’
2(l3) I5
as atTected by the sampling
September
of samples different
1979 to July
location
1980.
collected locations
No.
Central
Southern
of
samples
0
0 (0)’
18(?7)
4 (67)
4 (9) 21(49)
I4 (82)
cl000
3(18)
28 (43)
> 1000
2 (33) 6
I8 (42) 43
0 (0) I7
of samples
‘Percentage
of samples
shown
in parenthesis
20 (30) 66
Peanut rhizobia
In contrast to R. japonicum. Rhj~ob~um spp were found in many of the soil samples. These strains can nodulate many legumes such as peanut. Arachus hFpogaea. cowpea, Vigna Sirtensis lima bean, Phuseolus lunatus and others. Many of these plants are grown widely in Iraq. The absence or low numbers of Rhizobium spp found in the salt-affected soil samples (EC more than 4mmhos cm-‘) is consistent with the findings of Piliai and Sen (1966) on R. trifalii, Yadav and Vyas (1973) on R. melifuri, Steinborn and Roughley (1975) on R. [egttmi~osu~urn (nodulating peas), Ty (1981) on R. juponicttm and (A. N. Yousef, unpublished Ph.D. thesis, Dundee University, 1982) on R. feguminosarum nodulating field beans). The limited presence of Rhi:obitcm spp in the soil samples having more than I % organic carbon (Table 2) could be attributed to an effect, caused by the relatively high organic matter content, which activates other soil microflora such as fungi, actinomycetes and protozoa. One or more of these organisms might have an antagonistic effect on the rhizobial growth. Such effects, in fact have been reported by Damirgi and Johnson (1966). Moreover, during the decomposition of organic matter in the soil, some of the compounds released may either have a toxic effect on the bacteria or cause unfavourablc conditions for their growth and activity. According to Mulder and Van Veen (1960) there are unknown organic compounds that might have lethal effects on Rhixbittm in the soil. The fact that more than 85% of the populated samples had a pH value of 7.6-8. I could be explained by two masons. First, as soil bacteria, rhizobia in general, prefer to grow under neutral or slightly alkaline conditions. The second, is that such a range of pH is the most common one in Iraqi soils which is. therefore, expected to bc the favourable range for the occurrence of the indigenous microorganisms. The CEC of any soil varies with the amount and type of clay and organic matter. In the present study, although soil texture was not dctcrmincd. variations in the CEC could bc mainly attributed to the clay content of the soil samples since the organic carbon content was very low (86% of the whole samples contained <0.5-l % organic carbon, Table 3). Thcrefort, organic matter probably had no effect on the CEC value However. the effect of clay content on enhancing the survival of root-nodule bacteria have been documcntcd by Vyas and Prasad (1960) and Marshall (1968, 1975). Differences in the counts of Rlri-_ohilrnrspp, found under various cropping systems. could be due to various cultivation practices. According to Alexander (l96l), plowing and tillage operations are drastic environmental treatments that usually caused marked ba~tcriologi~l alterations. in addition, secretions of different plant-root systems may affect both type and density of soil microflora. Such effects, have been reported by Rovira and 1McDougall (1967). The evidence shown in Table 8 that the prcsencc of rhizobia was more affected during summer than in winter was cxpcctcd. In Iraq. mainly in the central and southern regions, the daily hig.h temperature along with the decrease in soil motsturc content, during summer months, could produce a partial
in Iraqi soils
395
ste~Iization effect in at least the upper IOcm soil layer. Similar findings have been also shown by Taylor and Burns (1924) and Abed-el-Malek and Rizk (1966). On the other hand low winter temperatures may only check bacterial activities but not destroy their cells. Therefore, the absence of Rhizobium spp from samples collected during December and January, might be a result of either a real absence of the bacteria or to a harmful effect of the preceding summer. To be certain there is any significant interactive effect between the peanut rhizobial number and some of the studied factors, the data were submitted to computer analysis using a multiple planer regression program. The correlation coefficients were: 0. I72 for the effect of data of sampling, geographical location and climatic factors, 0.318 for the effect of lime content and pH and 0.421 for electrical conductivity and cation-exchange capacity. Therefore, the only significant correfation was found between electrical conductivity and cation-exchange capacity (and rhizobial numbers) (N = 66, r = 0.36 and z = 0.1 Oh). The present investigation showed that, in Iraq. inoculation of soybean seeds with R. joponictmt is essential since the Rhizobium was not detected in any of the soil samples studied. Despite the fact that peanut rhizobia (Rlti:obt’trnt spp) were found in most of the samples, their occurrence and population were affected by several environmental factors. Since R~ti~i~bit~~tspp found here are indigenous, studies concerning their efficiency compared with other introduced strains, are of obvious importance for studies of symbiotic N,-fixation. REFERENCES Abed-el-Malek
Y. and Rizk S. G. (1966)
Microbial and soils. Jo~cmcrl of .tfkro1. 47-56. inrro&ction IO Soil Microhidc~~y.
nitrogen changes in Sharaqi bido~.v. ffttired Aruh Repubk
Alexander Wiley.
M.
( i 96 I )
New York.
Brockwcll 1. (1963) Accuracy of plant for counting Microbiology
population of 11, 377-383.
Rhixhm
infection tcchniqucs rr(/b/ii. App/ic*rl
Johnson H. W. (1966) Elkt of soil actinomycets on strains of Ritixhium jupcmirurtl. .J,qrrtt~onty Journnl 58, ‘23-226. Gibson A. H. (1976) Limitation to dinitrogcn fixation by legumes. Proceedings. Firsr In~~rn~~il~n~l S~~i~f).s;~~~~lott Nitrogen Fi.wtiwr (W. E. Newton and C. J. Nyman, Eds). pp. 4004128. Washington State University, Pullman,. Lie T. A. (1974) Environmental effects on nodulation and symbiotic nitrogen fixation. In Talc Bio/o& of ?V;irro,cytr Fi.wrion (A. Quispel. Ed.). pp. 555-582. North Holland. Amsterdam. Marshall K. C. (1968) Interaction between colloidal montmorillonite and cells of Rhizohium spccics w-ith different ionogenic surfaces. ~~~~~~z~#ri~~Bj(/p~f~.si~~iAcrcl 156, 179-168. Marshall K. C. (1975) Clay mineralogy in relation to survival of soil bacteria. Annual Rekw of Plryropurho/o~ty 13. 357-373. Mulder E. G. and Van Veen N. L. (1960) EKect of oH and organic compounds on nitrogen fixation by red’clover. Phnr and Soil 113, 91-l 13. Pillai R. N. and Sen A. (1966) Salt tolerance of R. rr@i. Indian Jmmd of Agriculture Stirnces 36, 80--8-t. Rovira A. D. and McDougall B. M. (1967) Microbiological and biochemical aspects of the rhizosphere. In %ii Damirgi S. M. and
396
A. N. YOCSEF er al.
Biochemistry (A. D. McLaren and G. F. Peterson. Eds). pp. 417-463. Dekker, New York. Steinborn J. and Roughley R. J. (I 975) Toxicity of sodium and chloride ions to Rhizobium spp in broth and peat cultures. Journal of Applied Bacteriology 39, 133-138. Taylor E. M. and Burns A. G. (1924) Soil temperatures during sharaqi period and their agricultural significance. Ministry of Agriculture, Egypt. Technical Bulletin No. 31. Tu J. C. (1981) Effect of salinity on Rhizobium root-hair interaction, nodulation and growth of soybean. Canadian Journal of Plant Sciences 61, 231-239. USDA Salinity Laboratory Staff (1954) Diagnosis and Improvemenr of Saline and Alkali Soils. Handbook No. 60, USDA, Washington.
Van Schreven D. C. (I 964) The effect of some actinomycetes on rhizobia and .4grobacrerium radiobacrer. Plant and Sod 21. 283-302. Vincent J. M. (1970) d Manualfor the Practical Stu