Influence of pesticides on the growth rate and plant ...

Pesticide Biochemistry and Physiology 84 (2006) 143–154 www.elsevier.com/locate/ypest

InXuence of pesticides on the growth rate and plant-growth promoting traits of Gluconacetobacter diazotrophicus M. Madhaiyan a,¤, S. Poonguzhali a, K. Hari b, V.S. Saravanan c, Tongmin Sa a a

b

Department of Agricultural Chemistry, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea Division of Crop Production, Microbiology lab, Sugarcane Breeding Institute, ICAR, Coimbatore 641007, Tamilnadu, India c Department of Biosciences, Vellore Institute of Technology, Vellore 632014, Tamilnadu, India Received 7 April 2005; accepted 28 June 2005 Available online 30 September 2005

Abstract The in vitro study to examine the eVect of diVerent pesticides on the growth and survival of Gluconacetobacter diazotrophicus strain PAL5 was carried out. Monocrotophos, Lindane and Dichlorvos had the most lethal actions against Gluconacetobacter grown on LGIP medium, while Endosulphan, Chlorpyriphos, and Malathion eVects were intermediate. Herbicides generally appeared to have no adverse eVect on the growth and survival of Gluconacetobacter in the medium except for the concentrations exceeding recommended rates. Fungicides, except Ridomil, had a slight eVect on the growth of Gluconacetobacter at recommended dose. With the various pesticides used, the cell morphology was aVected to a larger extent resulting in larger number of pleomorphic cells. Further their inXuence on the physiological traits like nitrogenase activity, indole-3-acetic acid, gibberellin A3 production, and phosphate and zinc solubilizing activity are discussed.  2005 Elsevier Inc. All rights reserved. Keywords: Pesticides; Nitrogenase activity; Indole-3-acetic acid; Gibberellic acid; Phosphate and zinc solubilization; Gluconacetobacter diazotrophicus

1. Introduction Sugarcane is a tropical plant and performs well under tropical conditions in the world. India is a tropical country with sugarcane cultivation from *

Corresponding author. Fax: +82 43 271 5921. E-mail address: [email protected] (M. Madhaiyan).

0048-3575/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2005.06.004

times immemorial. Today India has the privilege of being the largest producer of sugar from an area of 4.08 Mha with an annual production of 295.73 MT. Sugarcane, being an important commercial crop demands a massive input of nitrogen (N) and potassium (K) [1]. It has long been hypothesized that the apparent deWcit in N inputs is made up by biological nitrogen Wxation (BNF) [2,3]. There are

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M. Madhaiyan et al. / Pesticide Biochemistry and Physiology 84 (2006) 143–154

numerous reports on diazotrophs associated with sugarcane, since the isolation of Beijerinckia (B. Xuminense) from the surface of sugarcane roots by Dobereiner and Ruschel [4]. Unfortunately, many of the reports failed to establish the numbers and its relation to plant associated N-Wxing activity of the diazotrophs [5]. Gluconoacetobacter diazotrophicus a Gram-negative acid tolerant N-Wxing bacteria associated with sugarcane was reported Wrst by Cavalcante and Dobereiner [7]. This bacteria contributing abundant N to the crop [6] is of particular interest and unique from other diazotrophs for the following reasons: (i) it is able to Wx nitrogen in the presence of nitrate [7] and excrete almost half of the Wxed N in a form available to plants [8] (ii) inspite of its survival and growth within cane tissues, it does not easily infect intact sugarcane plants [9]. These characteristics of G. diazotrophicus, suggests its high potential in improving sugarcane N nutrition and productivity. Application of pesticides for improving crop productivity has become necessary in the present day agricultural practices, resulting in entry of these chemicals into soil and water ecosystems [10]. Amongst agricultural practices, the widespread use of plant protection products represents a potential threat to soil organisms [11], including diazotrophs resulting in either stimulatory or inhibitory eVects [12–16]. Most of the pesticides, being xenobiotic, are degraded by very few microorganisms [17–19], which developed the tendency to degrade them while they may be toxic for others. Extensive studies on the eVect of some commonly used pesticides on the soil have been well worked out on chemotrophic bacteria and cyanobacteria [20,21]. The eVect of some of the pesticides on beneWcial microorganisms—Azospirillum [16,22], Rhizobium [23–32], Bradyrhizobium [14,15,33], Cyanobacteria [20,21], Pseudomonads [34], diazotrophic purple nonsulphur bacteria [13], and Azotobacter [35] have been well studied. For the enhancement of crop growth and to maximize the yield of sugarcane, various formulations of pesticides are used but their inXuence on G. diazotrophicus remains uninvestigated. So the present study reports on: (a) the growth and survival G. diazotrophicus at recommended dose of diVer-

ent pesticides in a chemically deWned media, (b) the eVect of pesticides on cell morphology and physiological traits viz., nitrogenase activity, indole-3-acetic acid (IAA), and gibberellin A3 (GA3) production, phosphate (P) and zinc (Zn) solubilization of G. diazotrophicus.

2. Materials and methods 2.1. Strain and chemicals Gluconacetobacter diazotrophicus strain PAL5 ATCC49037 isolated from sugarcane roots [36] was kindly provided by Johanna Dobereiner (EMBRAPA, Itajai, RJ, Brazil). The pesticides used in this study are listed in Table 1 with the details of their chemical nature and they were of commercial grade. 2.2. Culture conditions Stock cultures of G. diazotrophicus strain PAL5 were maintained on LGIP [37] (g L¡1 composition: sucrose 100 (10%), K2HPO4 0.2, KH2PO4 0.6, MgSO4 · 7H2O 0.2, CaCl2 · 2H2O 0.02, Na2 MoO4 · 2H2O 0.002, FeCl3 · 6H2O 0.018, bromothymol blue (BTB): 0.5% in 0.2 M KOH 5 mL, pH 5.5) and potato agar slants (g L¡1 composition: potato tubers (peeled): 250, sucrose: 200 (20%), pH 5.5) as described by Cavalcante and Dobereiner [7] and transferred regularly. Inoculum was always added in a quantity suYcient to reach a concentration of 1% (v/v) in the culture medium. Treated bacteria were grown with 14 pesticides commonly used in sugarcane Welds, in the culture medium from the beginning of incubation. The dose of added pesticides is equivalent to that recommended for Weld conditions. The respective concentrations of pesticides prepared by appropriate dilutions in precooled double-distilled water were Wlter sterilized through a Millipore membrane Wlter (0.2 m) before their addition. For the nitrogenase assay culture grown in Nfb medium [38] (g L¡1 composition: malic acid: 5, K2HPO4: 0.5, MgSO4 · 7H2O: 0.2, NaCl: 0.1, KOH: 4.5, CaCl2: 0.02, Fe EDTA: 0.066, Na2MoO4 · 2H2O: 0.4 mg, MnSO4 · H2O: 0.47 mg, H3BO3: 0.56 mg, biotin/pyridoxal solution 1 mL;


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Table 1 List of tested pesticides in this study Type

Common name

Chemical name

Molecular formula

Chemical group

IC

Monocrotophos 36 EC

C7H14NO5P

Organophosphate

IC

Malathion 50% EC

C10H19O6PS2

Organophosphate

IC

Chlorpyriphos 20% EC

C9H11Cl3NO 3PS

Organophosphate

IC IC IC

Dichlorvos 76% EC Lindane 20% EC Endosulphan 35% EC

C4H7Cl2O4P C6H6Cl6 C9H6Cl6O3S

Organophosphate Organochlorine Organochlorine

HC

Butachlor 50% EC

C17H26ClNO 2

Chloroacetamides

HC

Alachlor 50% WP

C14H20ClNO 2

Chloroacetamides

HC

Atrazine 50% WP

C8H14ClN5

Triazines

HC FC FC

2,4-D 50% WP Bavistin 50 WP Ridomil MZ 72 WP

C8H5Cl2NaO5 C9H9N3O2 C15H21NO4

Phenoxy-carboxylic-acids Benzimidazole Phenylamide

FC

Dithane M45 75 WP Hinosan 50% EC

C4H6MnN2S4 · C4H6N2S4Zn C14H15O2PS2

Dithiocarbamate

FC

Dimethyl (E)-1-methyl-2-(methylcarbamoyl)vinyl phosphate S-1,2-bis(ethoxycarbonyl)ethyl-O, O-dimethyl phosphorodithioate O-O-Diethyl-O-3,5,6 trichloro 2-pyridyl phosphoro thioate Dimethyl 2-dichlorovinyl phosphate 1,2,3,4,5,6-hexachloro cyclohexane --1,2,3,4,7-Hexachloro bicyclo 2,2,1)-hepten-2-bisoxymethylen-5,6-sulphite N-Butoxymethyl-2-chloro-N-2,6diethylphenyl acetamide 2-Chloro 21-61 diethyl N-methoxy methyl acetanilide 6-Chloro-N2-ethyl-N4-isopropyl-1,3, 5-triazine-2,4-diamine 2,4-Dichlorophenoxyacetic acid Methyl benzimidazol-2yl carbamate 2[(2,6-Dimethylphenyl)-methoxyacetylamino]propionic acid methyl ester Zinc-manganese-ethylene-bis-dithio carbomate O-Ethyl-S-S-diphenyl-dithiophosphate

Organophosphate

IC, insecticide; HC, herbicide; and FC, fungicide.

BTB 0.01 g; pH 5.8) amended with 1.25 g NH4Cl was used. 2.3. Growth of G. diazotrophicus on diVerent pesticides Gluconacetobacter diazotrophicus strain PAL5 cells grown in LGIP harvested by centrifugation (3000g, 5 min), were washed three times with sterilized double-distilled water and dispensed equally in 50 mL LGIP in a series of 250 mL conical Xasks. Graded concentrations (the recommended Weld dose and 2-fold of recommended dose) of Wltersterilized pesticides prepared as mentioned above were added to the media before inoculating the culture. Cultures without pesticides served as the control. After 72 h, aliquots were withdrawn in triplicates and growth assessed both by turbidometric and drop plate method [39] and the cell morphology was observed under phase contrast microscope (DS-5M-L1 Nikon microscope, USA). The 50% inhibitory growth concentrations for the

isolates was determined by monitoring survivability of cells on potato agar plates containing graded concentrations of pesticides. The percentage survival of the cultures was calculated by the method: No. of colonies on the pesticide treated plates/No. of colonies on the pesticide untreated plates £ 100 [21]. The concentration of the pesticides at which 50% of colonies survived as compared to untreated control culture was termed the IGC50 and the concentration at which no colonies survived (complete lysis) were considered the lethal concentration. The experiment was repeated three times and every time Wve sets of plates were taken for each pesticide concentration. 2.4. Acetylene reduction assay The eVect of diVerent pesticides on nitrogen Wxing eYciency of G. diazotrophicus was assessed by acetylene reduction assay as described by Hardy et al. [40]. Penicillin Xasks (100 mL) contain-

146


ing 30 mL of Nfb medium inoculated with about 1 mL of the 72 h Nfb cultures (6 £ 108 CFU mL¡1) were Wtted with rubber plugs tightened with metal cowls and then incubated for 7 days at 30 °C. A control without the addition of pesticides was maintained. One milliliter of air samples withdrawn from the Xasks and analyzed in a Gas Chromatograph (CP3800 GC, Varian, German) with a Porapak-Q 80/100—column operated isothermally at 70 °C, with nitrogen gas carrier and a Xame ionization detector measured the ethylene production and compared with pure ethylene as a standard. Studies were performed in triplicate. 2.5. QuantiWcation of IAA production Extraction and HPLC of G. diazotrophicus strain PAL5 cultures for IAA were performed according to the methods of Lee et al. [41] with slight modiWcations. Bacterial cultures (20 mL) were made cell free by centrifugation at 3000g and Wltration, and supernatants were extracted three times with ethyl acetate after adjusting the pH to 2.5. Five- to Wfteen microliter aliquots of the Wltered extracts were injected into a HPLC (Bondesil, Varian Prostar), Separon Six C18—column (5 m) equipped with a diVerential OE-308/1 UV detector absorbing at 280 nm. The IAA was eluted with 30% methanol (acidiWed with 3-Xuoroacetic acid to pH 3.0) at a Xow rate of 0.5 mL min¡1. Peak retention times were compared with those of chemically synthesized IAA (Sigma, USA) standard and quantiWed by comparison of peak areas. 2.6. QuantiWcation of gibberellins (GA3) production Cultures of G. diazotrophicus strain PAL5 grown in 100 mL of LGIP medium with and without pesticides at 30 °C and 80 rpm in an orbital shaker water bath at 5 days till stationary phase [as determined OD540 (biomass production) and colony forming units (CFU)] was used for gibberellins production assay. After incubation period, the cultures were sonicated for 20 min and centrifuged at 10,000g for 30 min. The supernatant acidiWed to pH 3.0, and partitioned four times with equal volumes of ethyl acetate fraction (which would con-

tained free GAs) was evaporated, diluted in 10% methanol and injected on C18 reverse phase HPLC. Elution was done at 2 mL min¡1, in a Prostar HPLC (Varian) with a 10–73% gradient of methanol in 1% acetic acid. Peak retention times were compared with those of chemically synthesized gibberellin A3 (GA3) (Sigma, USA) standard and quantiWed by comparison of peak areas. 2.7. Determination of phosphate and zinc solubilizing activity The plates with LGIP medium containing 0.1% insoluble compounds viz., tricalcium phosphate [Ca3(PO4)2]/zinc oxide (ZnO) with diVerent concentrations of the various pesticides were spot inoculated with the strain PAL5 and incubated at 28 °C for 3 days and bacterial growth was compared with the control plates containing no pesticides. After incubation, the diameter of the zone of solubilization was measured in mm. The development of a clear zone at inoculation site on the culture plates was noticed as an index of phosphate solubilization and measured. 2.8. Statistical methods The data were subjected to statistical analysis and signiWcant diVerence was calculated at P 6 0.05 using SAS package, Version 8.2 [42].

3. Results The survival of the cells of G. diazotrophicus PAL5 grown under chemically stressed condition was monitored by exposing the cultures to graded concentrations of pesticides. G. diazotrophicus grown in LGIP medium, i.e., control without the addition of pesticides attained a maximum population of 10.2 Log No. of viable cells mL¡1 at 60 h. But the growth was reduced when treated with the recommended dose of insecticides and fungicides with further reduction in increasing the concentration of the added pesticides to 2-fold of the recommended dose. The insecticide Lindane had the most deleterious eVect with complete lysis of the cells at the recommended dose of 5 mg L¡1. The


147

Fig. 1. Variation of G. diazotrophicus strain PAL5 population in culture media amended with diVerent concentrations of insecticides. (A) Monocrotophos 36 EC; (B) Malathion 50% EC; (C) Chlorpyriphos 20% EC; (D) Dichlorvos 76% EC; (E) Lindane 20% EC; and (F) Endosulphan 35% EC.

inhibition was minimized with Dichlorvos (DDVP), Malathion, and Endosulfan with the population of G. diazotrophicus equaling control at the end of incubation (Fig. 1). Herbicides with the exception of 2,4-D had a meager eVect on the population when compared to insecticides and fungicides. The herbicides Butachlor, Alachlor, and Atrazine stimulated the growth of G. diazotrophicus at certain periods when they were added at

their recommended concentrations. But when the amounts added to the growth media increased, the population was slightly reduced. The herbicide 2,4D showed 50% inhibition of the cells at 22 mg L¡1, but independent of the amount added to the growth medium (Fig. 2). Dithane was the most deleterious among the fungicides followed by Ridomil. Bavistin was the least deleterious at the recommended dose (2.0 mg L¡1). With Dithane and

148


Fig. 2. Variation of G. diazotrophicus strain PAL5 population in culture media amended with diVerent concentrations of herbicides (A–D) and fungicides (E–H). (A) Butachlor 50% EC; (B) Alachlor 50% WP; (C) Atrazine 50% WP; (D) 2,4-D 50% WP; (E) Bavistin 50 WP; (F) Ridomil MZ 72 WP; (G) Dithane M45 75 WP; and (H) Hinosan 50% EC.


149

Fig. 3. The IGC50 values and recommended doses of diVerent pesticides used to study their eVects on the growth and survival of G. diazotrophicus strain PAL5. MC, Monocrotophos 36 EC; ML, Malathion 50% EC; CP, Chlorpyriphos 20% EC; DL, Dichlorvos 76% EC; LD, Lindane 20% EC; ES, Endosulphan 35% EC; BC, Butachlor 50% EC; AC, Alachlor 50% WP; AZ, Atrazine 50% WP; 2,4-D, 2,4-D 50% WP; BV, Bavistin 50 WP; RM, Ridomil MZ 72 WP; DE, Dithane M45 75 WP; and HS, Hinosan 50% EC.

Hinosan, the inhibition on the growth of G. diazotrophicus showed no signiWcant diVerences with the variations of the added doses to the growth media. The above results were conWrmed with the calculations of 50% growth inhibition value (IGC50) for the pesticides. The insecticide Lindane had its IGC50 value (2.5 g mL¡1) far below the recommended dose (5.0 g mL¡1). Complete inhibition of growth was observed with Monochrotophos, Dichlorvos, and Dithane when added at the rate of 6.0, 2.8, and 4.0 g mL¡1, respectively, with their IGC50 value equaling the recommended dose (Fig. 3). In contradictory, the other insecticides Malathion, Chlorpyriphos, and Endosulfan with minimum inhibition on growth of G. diazotrophicus had their IGC50 values greater than that of recommended dose and similar was the case with herbicides and fungicides. The observations of the cell under phase contrast microscope at 60 h of growth revealed diVerential morphological inXuences by the pesticides (data not shown). The insecticides and fungicides aVected the cell morphology to a greater extent compared to herbicides. The proportionate increase of deformities in cells excepting Atrazine could be positively correlated with the IGC50 values of the pesticides.

Biological Wxation of atmospheric dinitrogen by its reduction and protonation to Weld ammonia is achieved in diazotrophic bacteria by the enzyme nitrogenase [43]. So the proportionate inhibitions on the growth promoting aspects of G. diazotrophicus viz., nitrogenase activity and phytohormone production by the pesticides were studied (Table 2). Addition of pesticides to the growth media substantially reduced the nitrogenase activity of pure cultures of G. diazotrophicus. The percentage of inhibition in the nitrogenase activity ranged from a minimum (22.8%) to complete inhibition with no activity for the pesticides used. Similar trend was noticed for IAA and GA3 production also. Butachlor and Endosulfan with the percentage of inhibition being 22.8 and 33.4 were the least inhibitory. In contrast Monochrotophos, Dichlorvos, Lindane, and Ridomil totally inhibited the nitrogenase activity in G. diazotrophicus and Chlorpyriphos was most potent with 83.3% inhibition. Still, Monochrotophos retained a minimum of 22.4 and 8.6% of IAA and GA3 production. To the contrast Lindane and Ridomil had a complete inhibitory eVect on both IAA and GA3 production of G. diazotrophicus. But the percentage of inhibition, from 39.5 and 36.1 to complete inhibition for IAA and GA3,

150


Table 2 EVect of diVerent pesticides on nitrogenase activity, growth hormone production and P and Zn- solubilizing ability by G. diazotrophicus strain PAL5 Trade name

IAA (g mL¡1) Nitrogenase activity ¡1 ¡1 (nmol C2H4 h vial )

Monocrotophos 36 EC Malathion 50% EC Chlorpyriphos 20% EC Dichlorvos 76% EC Lindane 20% EC Endosulphan 35% EC Butachlor 50% EC Alachlor 50% WP Atrazine 50% WP 2,4-D 50% WP Bavistin 50 WP Ridomil MZ 72 WP Dithane M45 75 WP Hinosan 50% EC Control LSD (P 6 0.05)

— 13.0 § 1.2ed (80.9)* 11.4 § 1.0ed (83.3) — — 45.3 § 1.7cb (33.4) 52.5 § 3.3b (22.8) 17.9 § 1.2ed (73.6) 23.6 § 2.4d (65.3) 27.4 § 3.1cd (59.8) 42.6 § 3.5cb (37.4) — 12.0 § 0.7ed (82.3) 20.4 § 2.4d (70.0) 68.0 § 7.4a (00) 18.04

0.63 § 0.0efg (77.6) 0.84 § 0.02hfg (70.1) 0.87 § 0.08hfg (69.0) 0.19 § 0.03i (93.2) — 0.62 § 0.06h (77.9) 1.30 § 0.09ed (53.7) 1.61 § 0.17cb (42.7) 1.40 § 0.08cd (50.2) 1.70 § 0.22b (39.5) 1.10 § 0.06ef (60.9) — 0.80 § 0.0hg (71.5) 0.97 § 0.04efg (65.5) 2.81 § 0.13a (00) 0.32

GA3 (ng mL¡1) 5.0 § 0.4e (91.4) 35.0 § 1.8b (39.7) 13.2 § 0.4e (77.3) 2.01 § 0.1gf (96.5) — 15.3 § 1.3ed (73.7) 37.1 § 1.3b (36.1) 24.2 § 0.9cd (58.4) 27.1 § 1.2cb (53.4) 12.3 § 0.4e (78.8) 12.3 § 0.5e (78.8) — 11.3 § 0.9ef (80.6) 13.1 § 0.9e (77.4) 58.1 § 1.3a (00) 9.78

Solubilizing zone (mm) Ca3(PO4)2

ZnO

6.0 § 0.5c (77.8) 14.0 § 0.9b (48.2) — — — — 11.0 § 0.9cb (59.3) 14.0 § 0.5b (48.2) 14.0 § 0.9b (48.2) 14.0 § 1.4cb (48.2) 13.0 § 0.9b (51.9) — 12.0 § 1.4cb (5.6) — 27.0 § 0.9a (00) 3.94

5.0 § 0.7d (73.7) 11.0 § 0.5b (42.1) — — — — 4.0 § 0.9e (79.0) 7.0 § 0.9dc (63.2) 8.0 § 0.5dc (57.9) 9.0 § 0.5c (52.6) 11.0 § 0.9b (42.1) — 6.0 § 0.5de (68.4) — 19.0 § 0.9a (00) 2.07

Each value represents mean § SE of three replicates. In the same column, signiWcant diVerences at P 6 0.05 levels are indicated by diVerent alphabets. Data followed by same alphabet in the same column are not signiWcantly diVerent from each other. ¤, % inhibition compared to control; —, ND not detected (100% inhibition)/no solubilization zone occurred.

respectively, was comparatively higher than that of nitrogenase activity. Dichlorvos was most potent in inhibiting both IAA and GA3 production. The P and Zn solubilizing capacity of G. diazotrophicus with the addition of pesticides in the growth medium at a recommended dose revealed a complete diVerent scenario (Table 2). Most of the insecticides viz., Chlorpyriphos, Dichlorvos, Lindane, and Endosulfan and the fungicides Ridomil, and Hinosan were complete inhibitors of P and Zn solubilization. Hinosan with an IGC50 value higher (2.2 mg L¡1) than the recommended dose (2.0 mg L¡1) portraying no morphological eVects on the cells retained 30% of the growth promoting eVects of G. diazotrophicus but had a complete inhibition of P and Zn solubilization.

4. Discussion The increasing cost of chemical fertilizers and the concern of environmental pollution emphasize the importance of BNF in plant N nutrition. G. diazotrophicus is found to be a promising endo-

phytic N Wxer associated with sugarcane crop. After its discovery from Brazilian sugarcane varieties by Cavalcante and Dobereiner [7], it is being subsequently reported from other regions and various crops such as coVee [44,47], pineapple [45], and ragi [46]. Also its presence in various tropical and subtropical crops like carrot, raddish, and beetroot has been reported recently [47]. Persistant pesticides are likely to interact with soil and rhizosphere microorganisms [48] and Pham et al. [49] reported pesticides may cause speciWc stresses in bacterial cells related to DNA damage, protein damage, oxidative damage, or membrane damage. The pesticides inXuence on the activities of Azospirillum has been studied both in pure culture and in mixed populations [50,51]. On the present study investigated, the pesticidal eVects on the survival and growth promoting activities of G. diazotrophicus in a chemically deWned media. Variations could be seen in the eVects of pesticides with the type and nature of the chemicals used. With the diVerent chemicals used, herbicides with the exception of 2,4-D had a meager eVect on the population when compared to insecticides and


fungicides. But Singh et al. [21] reported reduced growth and chlorophyll content in the cyanobacterial isolates when treated with diVerent herbicides. But, herbicides in general appear to have no adverse eVects on the population of soil bacteria except for exceeding concentrations of the recommended rates [48]. The herbicide 2,4-D showed 50% inhibition of the cells at 22 mg L¡1 exhibiting a maximum inhibition among the herbicides. But the inhibition eVect of this herbicide was independent of the amount added to the growth medium. Previous studies also conWrm the varying eVects of the pesticides on microorganisms. The fungicide Mancozeb reduced the Bradyrhizobium sp. growth by 50% and aVected the bacteria peanut interaction [32]. The normal cells of G. diazotrophicus are straight rods with rounded ends and seen as single, pair or chain-like structures without endospores [52]. But, the observations of the cell under phase contrast microscope at 60 h of growth in present study revealed diVerential morphological inXuences by the pesticides. Pleomorphism in G. diazotrophicus was noticed under high N concentrations [53] and they further suggested that formation of pleomorphic cells would reduce the culturability of this bacterium in the high N fertilized Welds. Fabra et al. [15] reported changes in chemical composition of Bradyrhizobium sp. USDA3187 and attributed the cause to the alterations in cellular growth and nitrogen Wxation reported previously [32]. Addition of pesticides to the growth media also resulted in reduced nitrogenase activities of pure cultures of G. diazotrophicus and also signiWcantly reduced the plant growth hormones IAA and GA3 produced by G. diazotrophicus. Franke et al. [54] determined and found the nifH gene sequence of G. diazotrophicus to be clustered with Azospirillum brasilense, Rhodobacter capsulatus and Rhodospirillum ruburum and the pair wise sequence had 89.4% similarity with A. brasilense. The variations in the pesticidal eVects were also noticed in Azospirillum when bromoporpylate (acaricide) amended media had no eVect on the nitrogenase activity whereas in methidathion (insecticide) amended media there was a drastic reduction [16]. They further showed that the nitrogenase activity of the microorganisms would be

151

always varying under the presence of a pesticide. The pesticide Monochrotophos showed 44 and 46% inhibition on the nitrogenase activity of anoxygenic phototrophic non sulfur bacteria Rhodobacter spheroids and Rhodopseudomonas palustris [13]. Recent studies suggest that any beneWcial eVect that G. diazotrophicus may have on plant growth are more likely to be via mechanisms other than N2 Wxation such as production of IAA [55–57] and the amount of N2 Wxed by microorganisms is not enough to explain the total increase in N content in the inoculated plants [58]. It may be attributed to the ability of these organisms to augment the root system through mechanisms such as production of phytohormonal substances like auxins and gibberellins (GAs) [38,59]. Bastian et al. [60] reported the production of the gibberellins GA1 and GA3 by the cultures of G. diazotrophicus and has quantiWed them using isotope dilution analysis. In this study, an attempt was made to study the eVect of pesticides on IAA and GA3 production of G. diazotrophicus. SigniWcant reductions in the amount of IAA and GA3 produced by the pure cultures could be noted with the additions of pesticides. But still, some pesticides which completely inhibited nitrogenase activity were able to retain a minimum amount of IAA and GA3 produced. A plant growth promoting substance such as IAA known to be produced by G. diazotrophicus could be a nitrogen Wxation independent factor [41]. The acid production by G. diazotrophicus has been an added value, where it could also solubilize insoluble phosphates in broth assays [52]. G. diazotrophicus is able to solubilize the insoluble P and Zn compounds added in the media under both plate and broth assays [47]. Addition of pesticides to the culture media was found to be detrimental to the growth and activities of G. diazotrophicus and it emphasize that appropriate care should be taken in selecting a pesticide before its use. Although the results are convincing, it has to be stated that the experiments are performed under laboratory conditions, which still diVer from the natural environment. Nevertheless, the application methods and the degradation of the pesticides in natural environment play a key role in determining the actual concentrations available in soil [61].

152


Therefore, the results from the laboratory experiments must be conWrmed by additional experiments under natural conditions using Weld experiments.

Acknowledgments We thank J. Dobereiner, EMBRAPA, Itajai, RJ, Brazil for providing the type strain of G. diazotrophicus PAL5. The authors also thank Korea Science and Engineering Foundation for Wnancial assistance through the Research Centre for the Development of Advanced Horticultural Technology at Chungbuk National University.

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