192 M.H. Abd-Alla et al. Applied Soil Ecology 14 2000 191–200
AM. An increase in P nutrition and a resulting increase in N
2
fixation has also been suggested to account for the additional benefits of the synergistic relationship
Brown and Bethlenfalvay, 1987. Pesticides are essential for controlling plant pests,
and accordingly, improve the productivity of major crops including legumes. Pesticides applied to legu-
minous crops constitute a potential hazard to growth, nodulation and nitrogen accumulation Abd-Alla and
Omar, 1993. Nemec 1985 reported that nematicides had little or no effect on AM fungi, but Sreenivasa and
Bagyaraj 1989 reported that pesticides were toxic to AM fungi even at recommended levels. Menge 1982
reported that systemic fungicides have a more harmful effect on AM fungi than on non-systemic fungicides.
The effects of pesticide application on dual symbio- sis in temperate and tropical soils has been studied ex-
tensively e.g. Hetrick and Wilson, 1991; Sugavanam et al., 1994. However, the effect of pesticides on these
organisms and plant growth in desert soil is yet to be examined adequately. The objective of the current
study was therefore to examine the effects of pesticide application on AM–legume–rhizobiabradyrhizobia
associations in calcareous sandy soil.
2. Materials and methods
2.1. Plant growth Surface-sterilized seeds five seeds per pot of
common bean P. vulgaris L. cv. Kentucky Wonder, cowpea Vigna sinensis L. cv. Azmerly, and lupin
Lupinus albus L. cv. Marita were sown in plastic pots 28 cm diameter, 38 cm depth containing 5 kg of
sterilized calcareous sandy clay soil. The sterilization was carried out by heating the soil in metal buckets at
200
◦
C for 24 h. The physical and chemical character- istics of the soil are shown in Table 1. Available Ca,
Mg, and Na were determined according to Lanyon and Heald 1982, and soil P according to Olsen and
Sommers 1982.
Each pot was inoculated with 5 ml of appropriate rhizobia or bradyrhizobia suspension containing 10
7
cells ml
− 1
, prepared from 4-day old cultures grown in nutrient broth and added below the soil surface.
Rhizobium leguminosarium bv. phaseoli TAL 182, Bradyrhizobium cowpea CB 1015, and Bradyrhizo-
Table 1 Physical and chemical characteristics of the soil used
Property Value
Sand 55
Silt 12
Clay 23
CaCO
3
19 Electrical conductivity dS m
− 1
1.8 pH 1.2 soil:water
7.7 Organic matter
0.36 Total N
0.02 Exchangeable cation mg kg
− 1
Ca
2+
106 Mg
2+
28 Na
+
75
bium lupin WPBS 3201 D were used for inoculat- ing common bean, cowpea, and lupin, respectively.
Spores of Glomus fasciculatum Hall and Fish, 1979; Trappe, 1982 were isolated from the soil using a wet
sieving technique Gerdemann and Nicolson, 1963 and multiplied on onion roots Omar, 1995, 1998.
AM inoculum 50 gm of onion rhizosphere soil was added 2–3 cm below the soil surface. Ten days after
emergence, seedlings were thinned to three plants per pot. Plants were grown under greenhouse conditions.
Temperatures during the experimental period aver- aged 2812 daynight. Dilute 0.25 ionic strength,
Somasegaran and Hoben, 1985 nitrogen-free nutrient solution was added once a week and the soil water
content was adjusted to 60 water-holding capacity of the soil with distilled water every 2 days.
2.2. Pesticide application Five pesticides were used in this investigation. The
fungicide Afugan is commonly known as Pyrazophos with 30 active ingredient a.i.. Chemically, it is
0,0-diethyl 0-6-ethoxycarbonyl-5-methylpyrazolo-
[1,5-a]pyrimidin 2-ylphosphorothioate. Two her- bicides were also used: Brominal Bromoxynil or
Labuctrill-25, a benzoic acid herbicide 3,5-dibromo- 4-hydroxybenzonitrile with 24 a.i. formulation
and Gramoxone Paraquat, a bipyridyl herbicide 1,1-dimethyl-4,4-bipyridylium dichloride in 20
a.i. formulation. Afugan, Brominal and Gramox- one were manufactured by Hoechst AG, Frankfurt,
Germany and Plant Protection Limited, Fernhurst, Haslemore,
Surrey, England,
respectively. The
M.H. Abd-Alla et al. Applied Soil Ecology 14 2000 191–200 193
insecticide Selecron Profenfos, an organophospho- rus pesticide 0-4-bromo-2-chlorophenyl0-ethyl S-
n-propyl-phosphorothioate, in 72 a.i. formulation produced by Ciba Geigy Limited, Basel, Switzerland
and Sumi Oil formulated product of Suimithion, 0,0-dimethyl
0-3-methyl-4-nitrophenylphosphoro- thiate in 8 a.i. formulation produced by Kafar
El-Zayat Pesticides and Chemicals Co., Egypt. Afu- gan, Bromnial, Gramoxone, Selecron and Sumi Oil
were applied at the rate of 3.0, 0.6, 0.75, 0.9 and 0.13 mg a.i. kg soil
− 1
, respectively. These concen- trations are equivalent to the recommended field dose.
Conversion of field application rates to milligrams of pesticide per kilogram of soil was calculated assum-
ing an even distribution of the pesticide in the plough layer. Pesticides were diluted with water and added to
the top layer of the soil with irrigation water 2 days after planting and pots not receiving pesticides were
used as control. Pots were arranged in a randomized block design with four replicates and left to grow un-
der the conditions described above Section 2.1 until harvesting.
2.3. Measurements Plants were harvested every 20 days up to 60 days
after sowing, with almost the entire root system in- tact. From each pot for each treatment, one plant
was assessed after each harvest. Plant replicates for each treatment were of near uniform size and canopy
shape. The shoots were severed at the soil level, dried at 70
◦
C for 48 h and weighed. Roots were carefully separated from the soil together with rhizosphere soil
particles. Root and soil from each individual pot were carefully wet-sieved to collect the spores Gerdemann
and Nicolson, 1963. The spores were kept refrig- erated and each sample was adjusted to a suitable
volume. Aliquots measuring 1 ml were spread on grid filter paper and counted using a dissecting microscope.
After spore sieving, the roots were washed in distilled water and the nodules on each root were counted.
After nodule counting, each root mass was divided into two equal parts. One-half of the root mass was dried
at 70
◦
C and weighed. This root mass when doubled represented the total dry mass of the original sample.
The other part of the roots was cut into short lengths 3–5 mm to estimate the proportion of root length
colonized with AM fungi after clearing the roots in 2.5 KOH at 90
◦
C and staining with trypan blue, 0.05 in lactophenol Phillips and Hayman, 1970.
Dry matter from 60-day old plants was ground and digested in a triple acid mixture, and plant tissue P was
determined according to Olsen and Sommers 1982. N was estimated by the Kjeldahl method Black et al.,
1965 and K was estimated by a flame photometric method David, 1962.
2.4. Statistical analysis Data were subjected to one-way analysis of
variances with four replicates using a computer pro- gramme PC-stat. Treatment means were compared
using the least significant difference LSD test when significant F values occurred.
3. Results
