Bull Environ Contam Toxicol (2011) 86:384–388

DOI 10.1007/s00128-011-0231-1

Effect of Pesticides on Plant Growth Promoting Traits

of Greengram-Symbiont, Bradyrhizobium sp. strain MRM6
Munees Ahemad • Mohammad Saghir Khan

Received: 11 December 2010 / Accepted: 17 February 2011 / Published online: 26 February 2011
Ó Springer Science+Business Media, LLC 2011

Abstract The aim of this study was to investigate the adversely affects the metabolic activities of soil microflora
toxicity of herbicides (metribuzin and glyphosate), insecti- leading to the losses in soil fertility (Ahemad et al. 2009).
cides (imidacloprid and thiamethoxam) and fungicides For example, the changes in aerobic bacteria, autotrophic
(hexaconazole, metalaxyl and kitazin) at the recommended nitrifiers, respiration and nitrification in soils treated with
and the higher dose rates on plant growth promoting activi- herbicide, cinosulfuron at 42 (field rate) and 4,200 lg/kg
ties of Bradyrhizobium sp. under in vitro conditions. The occurred one and 4 weeks after incubation under labora-
Bradyrhizobium sp. strain MRM6 was isolated from nodules tory conditions. Of the measured parameters, only nitrifi-
of greengram plants. Pesticide-concentration dependent cation was slightly inhibited by cinosulfuron application
progressive-decline was observed in plant growth promoting at the field rate. Cinosulfuron at 100 mg/L negatively
traits of the strain MRM6 apart from exo-polysaccharides affected the growth of aerobic bacteria, fungi and Azoto-
which increased consistently on increasing pesticide con- bacter strains under conditions similar to those of soil
centrations. Generally, the highest toxicity to plant growth environment (Allievi and Gigliotti 2001). Therefore,
promoting characteristics of the Bradyrhizobium sp. strain understanding the toxic effects of pesticides on rhizobia
MRM6 was observed when the strain MRM6 was grown and symbiotic N2-fixation is important in order to evade
with three times the recommended field rates of glyphosate, the possible damage caused by the pesticide application.
imidacloprid and hexaconazole. In other study, Wani et al. (2005) reported that insecticide
phorate (applied at 100 and 500 lg/mL) substantially
Keywords Bradyrhizobium
Pesticide
Plant growth reduced the indole acetic acid production by phosphate
promoting rhizobacteria
Toxicity
Tolerance
Greengram solubilizing bacteria belonging to genera Serratia, Pseu-
domonas and Bacillus, isolated from various rhizospheric
soils. In contrast, phosphate solubilizing activity of these
The industrial progress in recent times has led to the bacteria was marginally affected in the presence of pho-
production of diverse agrichemicals such as fertilizers, rate. Similarly, Dunfield et al. (2000) assessed the effects
pesticides, soil conditioners and phytohormones. These of captan and thiram (fungicides) at rates of 0.25–2 g a.i./
chemicals are applied extensively and indiscreetly to kg seed on the survival and phenotypic characteristics
optimize the food production. A major fraction of these of the Rhizobium leguminosarum bv. viceae strain C1.
plant protection agents especially the pesticides is accu- Captan and thiram significantly reduced the numbers of
mulated in soils due to the repeated use and consequently, rhizobia recovered from seeds and altered the FAME and
Biolog profiles of recovered rhizobia. However, only the
highest concentrations of captan affected nodulation and
the legume growth. Contact with some seed-applied fun-
M. Ahemad
M. S. Khan (&) gicides can also significantly change the phenotypic
Department of Agricultural Microbiology,
characteristics of rhizobia. Therefore, from a range of
Faculty of Agricultural Sciences,
Aligarh Muslim University, Aligarh, U.P. 202002, India toxic pesticides, pesticides with fewer or without toxicity
e-mail: khanms17@rediffmail.com should be selected for further use.

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Bull Environ Contam Toxicol (2011) 86:384–388 385

Studies on the effect of various pesticides have largely culture (108 cells/mL) of bacterial isolates and was incu-
been focused on changes in diversity of soil microorgan- bated for 7 days at 28 ± 2°C with shaking at 125 rpm.
isms while the information concerning the impact of Indole acetic acid concentration in the supernatant was
agrochemicals on plant growth promoting activities of determined by the method of Gordon and Weber (1951),
bradyrhizobia is poorly investigated. later modified by Brick et al. (1991). The bradyrhizobial
The present study was therefore designed to evaluate the strains were also tested for siderophore [salicylic acid
effects of herbicides (metribuzin and glyphosate), insecti- and 2,3-dihydroxybenzoic acid] production using chrome
cides (imidacloprid and thiamethoxam) and fungicides azurol S agar medium and Modi medium supplemented
(hexaconazole, metalaxyl and kitazin) at the recommended, with 0, 19, 29 and 39 of pesticides following the method
double and three times the recommended field rates on the of Alexander and Zuberer (1991) and Reeves et al. (1983),
survival and in vitro plant growth promoting activities of respectively. Hydrogen cyanide (Bakker and Schipper
Bradyrhizobium sp. 1987), ammonia (Dye 1962) and exo-polysaccharides
(Mody et al. 1989) production by bradyrhizobial strains
was also determined.
Materials and Methods Each experiment was replicated three times using the
same treatments. The difference among treatment means
A total of 50 rhizobial strains were isolated from root was compared by honestly significant difference (HSD)
nodules of greengram [Vigna radiata (L.) Wilczek] plants using Tukey test at 5% probability level.
grown in experimental fields of Faculty of Agricultural
Sciences, Aligarh Muslim University, Aligarh (27° 290
latitude and 72° 290 longitude), India using yeast extract Results and Discussion
mannitol (YEM) medium (gL-1: mannitol 10; K2HPO4
0.5; MgSO4
7H2O 0.2; NaCl 0.1; yeast extract 1; CaCO3 A total of 50 rhizobial isolates recovered from the nodules
1; pH 7) (Vincent 1970). The experimental soil was an of greengram were tested to assess their tolerance to her-
alluvial sandy clay loam (sand 667 g kg-1, silt 190 g kg-1, bicides, glyphosate and metribuzin; insecticides, imida-
clay143 g kg-1, organic matter 6.2 g kg-1, Kjeldahl N cloprid and thiamethoxam, and fungicides, hexaconazole,
0.75 g kg-1, Olsen P 16 mg kg-1, pH 7.2 and water metalaxyl and kitazin (Table 1) on minimal salt agar
holding capacity 0.44 ml g-1, cation exchange capac- medium. Of these, strain MRM6 was Gram negative rods
ity 11.7 cmol kg-1 and 5.1 cmol kg-1 anion exchange and showed a variable biochemical reaction (Table 2). The
capacity). The rhizobial strains were characterized and rhizobial strain MRM6 was identified as Bradyrhizobium
identified as Bradyrhizobium sp. following Bergey’s sp. on the basis of nodulation test. Strain MRM6 had the
Manual of Determinative Bacteriology (Holt et al. 1994) highest MTL to all herbicides, insecticides and fungicides
and nodulation test using greengram as the host plant in and produced the plant growth promoting substances like,
sterile soils (Somasegaran and Hoben 1994). The brady- siderophores, indole acetic acid, exo-polysaccharides,
rhizobial strains were tested for sensitivity/tolerance to hydrogen cyanide and ammonia, in substantial amounts
metribuzin, glyphosate, imidacloprid, thiamethoxam, hex- (Table 3). In addition, the tolerance level of the strain
aconazole, metalaxyl and kitazin, by agar plate dilution MRM6 against the regularly increasing concentration of
method using minimal salt agar medium (g l-1: KH2PO4 1, each pesticide in minimal salt agar medium was consid-
K2HPO4 1, NH4NO3 1, MgSO4
7H2O 0.2, CaCl2
2H2O erably higher (Table 3).
0.02, FeSO4
7H2O 0.01, pH 6.5). The freshly prepared agar This study demonstrated that the Bradyrhizobium sp.
plates were amended separately with increasing concen- strain MRM6 had a wide-range of tolerance (MTL:
trations (0–3200 lg mL-1; at a two-fold dilution interval) 1,800–3,200 lg mL-1) towards the selected pesticides.
of pesticides. Plates were spot inoculated with 10 ll of The tolerance or resistance among microorganisms against
108 cells mL-1 bradyrhizobial strains and incubated at pesticides is a complex process, regulated at physiological
28 ± 2°C for 72 h. The highest concentration of each and genetic level. And hence, the microorganisms that
pesticide supporting bradyrhizobial growth was defined as develop resistance to a pesticide are capable of frequently
the maximum tolerance level (MTL). degrading it (Kumar et al. 1996; Ortiz-Hernández and
For quantitative assay of indole acetic acid, the brady- Sánchez-Salinas 2010). The temporary resistance (toler-
rhizobial strains were grown in Luria–Bertani (LB) broth. ance) against pesticides in general, is however, attributed to
Luria–Bertani broth (100 mL) containing fixed concentra- physiological changes that induce the microbial metabo-
tion of tryptophan (100 lg/mL) supplemented with 19, 29 lism leading to the formation of a new metabolic pathway
and 39 of the recommended rate of each pesticide and to bypass a biochemical reaction that could otherwise be
without pesticide (control) was inoculated with one ml inhibited by a specific pesticide (Bellinaso et al. 2003).

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Table 1 Pesticides used in the present study

Common name Grade (purity) Chemical name Chemical family Recommended
dose

Metribuzin (H) Commercial (70%w/w) 4-amino-6-tert-butyl-4,5-dihydro-3-methylthio- Triazinone 850 lg kg-1

1,2,4-triazin-5-one
Glyphosate (H) Commercial (71% w/w) N-(phosphonomethyl)glycine Organophosphate 1444 lg kg-1
Imidacloprid (I) Technical (100% EC) (E)-1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin- Pyridylmethylamine 100 lg L-1
2-ylideneamine
Thiamethoxam (I) Technical (100%w/w) (EZ)-3-(2-chloro-1,3-thiazol-5-ylmethyl)-5-methyl- Thiazole 25 lg L-1
1,3,5-oxadiazinan-4-ylidene(nitro)amine
Hexaconazole (F) Technical (100%w/w) (RS)-2-(2,4-dichlorophenyl)-1-(1H-1,2,4-triazol- Conazole 40 lg kg-1
1-yl)hexan-2-ol
Metalaxyl (F) Commercial (35%w/w) Methyl N-(methoxyacetyl)-N-(2,6-xylyl)-DL-alaninate Anilide 1500 lg kg-1
Kitazin (F) Commercial (48% EC) O,O-Bis(1-methylethyl) S-phenylmethyl Organophosphate 96 lg kg-1
phosphorothioate
H herbicides, I insecticides, F fungicides

Table 2 Morphological and biochemical characteristics of the substantially reduced the size of siderophore-zone. At the
Bradyrhizobium sp. strain MRM6 recommended rate, there was no inhibitory effect of any
Characteristics Strain MRM6 pesticide except hexaconazole and metalaxyl, which
reduced the siderophore-zone by 8%, compared to control.
Morphology
Maximum decline in the zone diameter was observed at the
Gram reaction -
39 of each pesticide (Table 3).
Shape Rods
The ethyl acetate extraction of the culture supernatant of
Biochemical reactions
the strain MRM6 grown in the Modi medium in the
Citrate utilization -
absence of pesticide produced 32 and 18 lg/mL salicylic
Indole ? acid and 2,3-dihydroxybenzoic acid. Pesticide-concentra-
Methyl red ? tion dependent progressive decline for both types of the
Nitrate reduction ? siderophores was observed. Decrease in salicylic acid and
Oxidase - 2,3-dihydroxybenzoic acid released by the Bradyrhizobium
Voges proskauer ? sp. strain MRM6 however, varied with the types of pesti-
Carbohydrate utilization cides. Among herbicides, glyphosate had a maximum
Dextrose - inhibitory effect on salicylic acid and 2,3-dihydroxyben-
Lactose - zoic acid. For instance, 39 of glyphosate decreased sali-
Mannitol ? cylic acid and 2,3-dihydroxybenzoic acid by 28% over
Sucrose - control. Imidacloprid at 39 decreased salicylic acid and
Hydrolysis 2,3-dihydroxybenzoic acid by 72 and 33%, respectively,
Starch ? while 39 of hexaconazole reduced salicylic acid and
Gelatin - 2,3-dihydroxybenzoic acid maximally by 37 and 39%,
? indicates positive and - indicates negative reactions respectively compared to the control. Among the selected
pesticides, hexaconazole at 39 generally, exhibited the
highest toxicity to salicylic acid and 2,3-dihydroxybenzoic
Permanent resistance, on the other hand, depends on genetic acid synthesis (Table 3). In the aerobic environment, iron
modifications, inherited by the subsequent generation of occurs principally as Fe3? and is likely to form insoluble
microbes (Herman et al. 2005; Johnsen et al. 2001). hydroxides and oxyhydroxides, thus making it generally
The siderophores-production by the pesticide-tolerant inaccessible to microorganisms. To acquire sufficient iron,
strain MRM6 was determined on the chrome azurol S agar the most common strategy adopted by bacteria is the
plates supplemented with varying concentration of each secretion of siderophores, low-molecular mass iron chela-
pesticide. The Bradyrhizobium sp. strain MRM6 revealed tors with high association constants for complexing iron.
the siderophores-producing ability by forming an orange Thus, siderophores act as solubilizing agents for iron from
colored zone of 13 mm on pesticide-free chrome azurol S minerals or organic compounds under conditions of iron
agar medium. Addition of pesticides to the medium limitation (Miethke and Marahiel 2007).

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In the absence of pesticide, the Bradyrhizobium sp. by directly stimulating plant cell elongation or cell division
strain MRM6 produced a maximum (38 lg/mL) amount of (Khan et al. 2010).
indole acetic acid, which however, decreased progressively In contrast to other plant growth regulating substances,
with increasing concentration of each pesticide. The toxic exo-polysaccharides produced by the strain MRM6
effect of pesticides on indole acetic acid was in the order: increased gradually with consistent increase in pesticide
hexaconazole [ glyphosate [ imidacloprid. Hexaconaz- dose. The greatest stimulatory effect on exo-polysaccharides
ole, glyphosate and imidacloprid at 39, decreased the production was recorded for glyphosate, which increased it
indole acetic acid by 42, 39 and 29%, respectively over by 10, 24 and 38% at 19, 29 and 39, respectively compared
untreated control (Table 3). The indole acetic acid so to the control (Table 3). Why exo-polysaccharides increased
produced by rhizobacteria is known to promote root growth on increasing pesticide-stress? The reason is unknown.

Table 3 Plant growth promoting activities of the Bradyrhizobium strain MRM6 in the presence of varying concentrations of pesticides
Pesticides Dose Plant growth promoting activities
(lg/L)
Siderophores EPSf Ammonia HCNg
(lg/mL)
Zone on CASa SAb DHBAc IAAd
agar (mm) (lg/mL) (lg/mL) (lg/mL) 100Te

Metribuzin 850 13 ± 1.5a 28 ± 1.2c 16 ± 0.9c 30 ± 2.6gh 21 ± 1.1 h

? ?
a d d j gh
1,700 13 ± 1.0 27 ± 2.1 15 ± 1.3 27 ± 1.5 22 ± 2.3 ? ?
2,550 13 ± 1.2a 24 ± 1.5gh 12 ± 1.2 g 26 ± 2.0jk 25 ± 1.4d ? ?
a bc c i f
Glyphosate 1,444 13 ± 1.6 29 ± 2.2 16 ± 1.4 28 ± 2.5 23 ± 0.9 ? ?
2,888 13 ± 1.4a 26 ± 1.7ef 14 ± 1.1e 25 ± 1.4 k
26 ± 1.5 cd
? ?
4,332 13 ± 0.8a 23 ± 2.4 h
13 ± 1.1 fg
23 ± 1.5 l
29 ± 1.6a ? ?
a c c ef h
Imidacloprid 100 13 ± 1.4 28 ± 1.5 16 ± 0.8 32 ± 2.2 21 ± 2.3 ? ?
200 13 ± 1.3a 25 ± 2.2f 14 ± 2.0e 30 ± 1.4gh 23 ± 2.4f ? ?
ab h g j d
300 12 ± 0.6 23 ± 1.5 12 ± 1.3 27 ± 1.5 25 ± 1.5 ? ?
Thiamethoxam 25 13 ± 1.3a 30 ± 1.3ab 17 ± 2.1bc 35 ± 1.2bc 22 ± 2.1gh ? ?
50 12 ± 1.2ab 29 ± 1.2bc 16 ± 1.8c 33 ± 2.2d 23 ± 1.5f ? ?
75 11 ± 1.0b 27 ± 1.5d 15 ± 1.3d 31 ± 1.7 fg
26 ± 2.3 cd
? ?
Hexaconazole 40 12 ± 1.0ab 26 ± 2.0ef 16 ± 2.4c 28 ± 2.1i 22 ± 2.1gh ? ?
b h e k d
80 11 ± 1.0 23 ± 2.3 14 ± 1.7 24 ± 1.6 25 ± 1.6 ? ?
120 11 ± 0.8b 20 ± 1.4i 11 ± 0.9 h
22 ± 1.4 l
27 ± 2.3b ? ?
Metalaxyl 1,500 12 ± 1.4ab 28 ± 1.3c 17 ± 1.3bc 35 ± 2.3bc 22 ± 0.9gh ? ?
3,000 12 ± 1.2ab 27 ± 2.2d 15 ± 2.1d 32 ± 1.4ef 24 ± 1.4ef ? ?
4,500 11 ± 0.9b 25 ± 1.4f 13 ± 1.7 fg
29 ± 1.6hi 26 ± 1.5 cd
? ?
Kitazin 96 13 ± 1.0a 30 ± 2.5ab 17 ± 2.2bc 36 ± 2.4b 21 ± 1.6 h ? ?
192 13 ± 1.2a 28 ± 2.1c 16 ± 2.6c 34 ± 2.1 cd 22 ± 1.3gh ? ?
288 13 ± 1.0a 26 ± 1.5ef 14 ± 1.8e 31 ± 1.7 fg
24 ± 2.4ef ? ?
a a a a h
Control (without pesticide) 13 ± 0.8 32 ± 1.8 18 ± 1.3 38 ± 1.8 21 ± 1.6 ? ?
LSD (p B 0.05) 0.57 1.15 1.09 1.19 1.10 - -
F value (treatment) 58.3 167.5 241.4 351.5 279.3 - -
Maximum tolerance levels (MTL) against metribuzin, glyphosate, imidacloprid, thiamethoxam, hexaconazole, metalaxyl and kitazin were 3,200,
3,000, 2,200, 2,400, 1,800, 2,800 and 3,200 lg mL-1. Values indicate mean of three replicates. Mean values (±S.D.) followed by different
letters are significantly different within a row or column at p B 0.05 according to Tukey test
a
Chrome azurol S agar
b
Salicylic acid
c
2,3 Dihydroxy benzoic acid
d
Indole acetic acid
e
Tryptophan concentration (lg ml-1)
f
Exopolysaccharides
g
Hydrogen cyanide; ? indicates positive reaction

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