Survival of bio-inoculants on fungicides-treated seeds of wheat,pea and chickpea and subsequent effect on chickpea yield

Survival of Mesorhizobium ciceri (SP₄) and Azotobacter chroococcum (CBD-15 and M₄) was tested on chickpea (Cicer arietinum) seeds treated with fungicides bavistin [methyl N-(1H-benzimidazol-2yl) carbamate] and thiram (tetramethyl-thiuram disulfide), whereas survival of phosphate solubilizing bacteria (PSB), Pseudomonas striata (27) and Bacillus polymyxa (H₅) was examined on two cultivars (Arkel and BV) of pea (Pisum sativum) seeds treated with thiram. Viability of Azotobacter chroococcum (W₅) was also examined on wheat (Triticum aestivum) seeds treated with bavistin, captan (cis-N-trichloromethyl thio-4 cyclohexane-1, 2-dicarboximide) and thiram under laboratory conditions using standard dilution and the plate count technique. All the tested strains of diazotrophs and PSB showed decline in their viable population on prolonged contact with fungicides. However, PSB showed variation in their viable population even with the cultivar.

BV cultivar of pea seeds showed better recovery of viable P. striata (10.75 to 10.61 log no. of viable cells with in 0-24 hrs) in the presence of thiram, whereas the Arkel cultivar of pea resulted in better recovery of viable B. polymyxa. Azotobacter chroococcum (W₅), a potential strain for wheat, showed better survival in the presence of bavistin, compared to thiram and captan. Higher viable population of Mesorhizobium ciceri (SP₄) and Azotobacter chroococcum (M₄) was recovered from chickpea seeds treated with bavistin compared to thiram. However, thiram-treated seeds resulted in a greater number of extractable Azotobacter chroococcum (CBD-15). Under field conditions, adverse effect of thiram was reflected on the performance of Mesorhizobium ciceri (SP₄) and A. chroococcum (M₄) strains, resulting in reduced root and shoot biomass and grain yield, compared to bavistin treated and culture inoculated treatment. CBD-15 showed better performance in the presence of thiram compared to bavistin.     

Fungicidal impact on chickpea--Mesorhizobium symbiosis

The effects of carbendazim, captan, thiram, and mancozeb, on plant vitality, chlorophyll content, N uptake, protein content, nodulation, and seed yield in chickpea (Cicer arietinun) were assessed in a controlled environment. Seeds treated with fungicides at 1 and 1.5 g.a.i. kg seed had no significant adverse effect on plant vigor, seed yield, and N and protein contents. In contrast, fungicides applied at 2 g.a.i./kg of captan, thiram and mancozeb, significantly reduced the measured parameters. In general, the toxicity of fungicides in terms of seed yield increased in the following order: Control=carbendazim > thiram > captan > mancozeb. Total chlorophyll content in foliage declined consistently with fungicides dose rates and application days. Seeds treated with lower rates of fungicides significantly increased nodulation (nodule number per plant and its dry mass) and were compatible with chickpea inoculum used in this study. Although carbendazim at 2 g.a.i. kg seed had no phytotoxic effect assessed under greenhouse conditions, it significantly reduced the chlorophyll content, nodulation (60d) and N content in shoots.     

Fungicidal Impact on Chickpea–Mesorhizobium Symbiosis

Abstract

The effects of carbendazim, captan, thiram, and mancozeb, on plant vitality, chlorophyll content, N uptake, protein content, nodulation, and seed yield in chickpea (Cicer arietinum) were assessed in a controlled environment. Seeds treated with fungicides at 1 and 1.5 g. a.i./kg seed had no significant adverse effect on plant vigor, seed yield, and N and protein contents. In contrast, fungicides applied at 2 g. a.i./kg of captan, thiram and mancozeb, significantly reduced the measured parameters. In general, the toxicity of fungicides in terms of seed yield increased in the following order: Control = carbendazim > thiram > captan > mancozeb. Total chlorophyll content in foliage declined consistently with fungicides dose rates and application days. Seeds treated with lower rates of fungicides significantly increased nodulation (nodule number per plant and its dry mass) and were compatible with chickpea inoculum used in this study. Although carbendazim at 2 g a.i./kg seed had no phytotoxic effect assessed under greenhouse conditions, it significantly reduced the chlorophyll content, nodulation (60 d) and N content in shoots.     

Tolerance to agricultural pesticides of strains belonging to four genera of Rhizobiaceae

In order to determine their tolerance to pesticides, 122 strains of rhizobia isolated from different geographical regions, and belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium and Bradyrhizobium were tested against eight herbicides, four fungicides and five insecticides. Sensitivity to the pesticides was measured by using the filter paper disk method at four concentrations, 0.45, 4.5, 45 and 450 μg per disk. When the pesticides were used at 0.45 μg per disk, a concentration similar to that found when pesticides are applied under field conditions, no inhibition was observed. Strains growth was affected at concentrations of 45 and 450 μg pesticide per disk. These higher concentrations can be encountered when seeds are treated with pesticides. Pesticides tolerance level was correlated to pesticide function, i.e rhizobial strains were more tolerant to insecticides, followed by herbicides and then fungicides. Two fungicides, captan and mancozeb, inhibited the highest number of strains. Only one insecticide, carbaryl, affected the growth of some rhizobial strains. Strains isolated from the arctic (Mesorhizobium spp. and R. leguminosarum bv. viciae), a putative pesticides-free environment, were either less or equally affected by pesticides compared to strains isolated from agricultural regions.     

Effects of trifluralin on soil microbial populations and the nitrogen fixation activities

Effects of trifluralin on soil microbial populations and the nitrogen fixation activity of nitrogen-fixing bacteria Azotobacter chroococcum and Bradyrhizobium japonicum and the decomposition of trifluralin by soil microorganisms were studied. Trifluralin at lower concentrations from 0.5 mg microg(-1) dry soil to lower than 10.0 mg microg(-1) dry soil appeared to stimulate the growth of soil bacteria, actinomycetes, mould, and the pure cultures of Br. japonicum and A. chroococcum. Not only the colony amounts of these two species of nitrogen-fixing bacteria increased, grown on agar medium containing lower concentrations of trifluralin, but also these colonies also enlarged in size and appeared obviously in shorter formation time. However, trifluralin at higher concentrations would inhibit the development of microbial colonies both in amount and size. Trifluralin inhibited the activity of acetylene reduction of A. chroococcum when it was added at the same time of inoculation with A. chroococcum, but it showed a noteworthy stimulation to nitrogen fixation of A.chroococcum when it was put into culture after the cells of the nitrogen-fixing bacterium had grown well. The observation that soil microorganisms could use trifluralin as sole carbon and nitrogen resources for their growth, indicated that microorganisms could decompose trifluralin well.     

Growth of Azotobacter chroococcum in chemically defined media containing p-hydroxybenzoic acid and protocatechuic acid

Growth and utilization of different phenolic acids present in olive mill wastewater (OMW) by Azotobacter chroococcum were studied in chemically defined media. Growth and utilization of phenolic acids were only detected when the microorganism was cultured on p-hydroxybenzoic acid at concentration from 0.01% to 0.5% (w/v) and protocatechuic acid at concentration from 0.01% to 0.3% (w/v) as sole carbon sources suggesting that only these phenolic compounds could be utilized as a carbon source by A. chroococcum. Moreover when culture media were added with a mixture of 0.3% of protocatechuic acid and 0.3% p-hydroxybenzoic acid, the microorganism degradated in first place protocatechuic acid and once the culture medium was depleted of this compound, the degradation of p-hydroxybenzoic acid commenced very fast.     

Residual behaviour and risk assessment of flubendiamide on chickpea (Cicer arietinum L.)

The study was undertaken to determine the disappearance trends of flubendiamide residues on chickpea under field conditions and thereby, ensure consumer safety. Average initial deposits of flubendiamide on chickpea pods were found to be 0.68 and 1.17 mg kg⁻¹, respectively, following three applications of flubendiamide 480SC @ 48 and 96 g a.i. ha⁻¹ at 7 d intervals. Half-life of flubendiamide on chickpea pods was observed to be 1.39 and 1.44 d, respectively, at single and double dosages whereas with respect to chickpea leaves, these values were found to be 0.77 and 0.86 d. Desiodo flubendiamide was not detected at 0.05 mg kg⁻¹ level on chickpea samples collected at different intervals. Theoretical maximum residue contribution (TMRC) for flubendiamide was calculated and found to be well below the maximum permissible intake (MPI) on chickpea pods and leaves at 0-day (1 h after spraying) for the both dosages. Thus, the application of flubendiamide at the recommended dose on chickpea presents no human health risks and is safe to the consumers.     

Development of controlled release formulations of thiram employing amphiphilic polymers and their bioefficacy evaluation in seed quality enhancement studies

Controlled release formulations of Thiram (Dimethylcarbamothioylsulfanyl-N,N-dimethylcarbamodithioate), a contact fungicide, have been prepared using laboratory synthesized poly(ethylene glycol) (PEG) based functionalized amphiphilic copolymers. The kinetics of thiram from developed controlled release (CR) formulations were studied in comparison with that of the commercially available 75 WS. Release from the commercial formulation was faster than with the developed CR formulations. Maximum amount of thiram was released on 35th day for PEG-2000 4d, 28th day for PEG-1500 4c, 21st day for PEG-1000 4b and 15th day for PEG-600 4a in comparison to commercial formulation (7th day). The diffusion exponent (n) of thiram in water ranged from 0.356 to 0.545 in the tested formulations. The half-release (t_1/2) values ranged between 14.78 to 22.1 days, and the Period of Optimum Availability (POA) of thiram ranged from 7.79 to 25.15 days. An effort has also been made to identify the suitable polymers that could reduce the seed deterioration during storage and also act as an effective carrier of fungicide thiram. The results demonstrate that the seeds coated with the different formulations deteriorated at a slower pace as manifested in high germination percentage over control. Apart from the fungicidal effect of thiram, the polymers acted as barriers to moisture reducing the rate of seed deterioration and checked the degradation of thiram. The CR formulation 4d, with PEG 2000, was found to be most effective as seed coat.     

Effect of aldicarb on the growth, respiration and (14C) - glucose metabolism of Axotobacter chroococcum BEIJ.

Aldicarb [2-methyl-2(methyl thio) propionaldehyde-O-methyl carbamoyl oxime], which forms the active ingredient of a systemic oximic carbamate insecticide, at 2 ppm level did not affect the in vitro growth and respiration of Azotobacter chroococcum Beij, while concentrations at 5 ppm and 10 ppm levels were inhibitory. The insecticide treatment at 10 ppm level suppressed the assimilation of (14C) - glucose in the whole cells and in the cellular constitutents viz., cold - TCA soluble, hot TCA soluble fractions and insoluble residue. However, the 14C - incorporation in the alcohol soluble and alcohol-ether soluble fractions was enhanced indicating that aldicarb considerably altered the glucose metabolism of the organism.     

In Vitro effects of various xenobiotics on Azotobacter chroococcum strains isolated from soils of southern Poland

Fourteen Azotobacter chroococcum strains isolated from soils of Southern Poland were studied concerning resistance to various xenobiotics: heavy metal ions: Cd(2+,) Cu(2+), Fe(3+), Mn(2+), Pb(2+), Zn(2+), pesticides: herbicides linuron (3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea) and combination of mecoprop ((RS)-2-(4-chloro-2-methylphenoxy)propanoic acid), dicamba (3,6-dichloro-2-methoxybenzoic acid) and MCPA (2-methyl-4-chlorophenoxyacetic acid), fungicide copper oxychloride, insecticide fenitrothion (O,O-Dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate) and eight antibiotics commonly used against Gram-negative bacteria. The tested soils were divided into seven groups of land use: forest, field crop, park, urban lawn, industrial area, garden and fallow land, and were analyzed for the following heavy metal ion concentrations using the atomic absorption spectrometry (AAS) technique: Cd(2+,) Cu(2+), Fe(3+), Mn(2+), Pb(2+), Zn(2+). All strains were resistant to Pb(2+), whereas other metals caused the growth inhibition of the analyzed strains. There was no significant relationship between metal concentrations in the analyzed soils and metal resistance of the isolates. Herbicide linuron did not inhibit the growth of A. chroococcum in any of the concentrations. All other pesticides caused the growth inhibition only in the concentrate forms. All isolates were sensitive to β-lactam antibiotic Meropenem, however high intraspecies differentiation was observed concerning resistance to other antibiotics. The obtained results require further study regarding resistance mechanisms and possible use of the xenobiotic-resistant strains in land rehabilitation.     

Influence of bacteria on Pb and Zn speciation, mobility and bioavailability in soil: A laboratory study

A soil column experiment was carried out to investigate the effects of inoculation of bacteria on metal bioavailability, mobility and potential leachability through single chemical extraction, consequential extraction and in situ soil solution extraction technologies. Results showed that bacteria inoculated, including Azotobacter chroococcum, Bacillus megaterium and Bacillus mucilaginosus, may pose both positive and negative impacts on bioavailability and mobility of heavy metals in soil, depending on the chemical nature of the metals. The activities of bacteria led to an increase of water dissolved organic carbon (DOC) concentration and a decrease of pH value, which enhanced metal mobility and bioavailability (e.g. an increase of water-soluble and HOAc-soluble Zn). On the other hand, bacteria could immobilize metals (e.g. a great reduction of water-soluble Pb) due to the adsorption by bacterial cell walls and possible sedimentation reactions with phosphate or other anions produced through bacterial metabolism.     

Residues and mutagenicity of captan applied to apple trees and potential human exposure

The fungicide captan (cis-N-((trichloromethyl)thio) 4-cyclo-hexene-1,2-dicarboximide) was applied at the rate of 2.4 g/l to apple trees (c.v. Golden Delicious) individually or as part of a standard treatment program where it was applied eight times during the growing season together with several pesticides. Leaf samples (100 discs of 2.2 cm diameter) were collected from treated and control trees before treatment and at 0, 1, 3, 7, 14, 28, 56, 90 and 112 days after treatment. Fruit samples were taken at mid-season (56 days) and at harvest (112 days). The objective of this study was to determine the captan residue and mutagenicity of leaf and fruit extracts to ascertain the potential health hazard to agricultural workers in these orchards. Surface residues were extracted from leaves and fruits with methylene chloride. These extracts were subsequently analyzed for captan by gas-liquid chromatography (GLC) utilizing an electron-capture detector, and for mutagenicity with two strains (TA98 and TA100) of Salmonella typhimurium, with and without microsomal enzyme activation. Positive mutagenic effects were observed with strain TA100 at 0-14 days post spray, even with extracts from one leaf disc's surface (3.8 cm2) of the single treatment. Captan residues in these samples indicated a decline from 9.3 micrograms/cm2 at 0 days to 0.80 micrograms/cm2 at 14 days and a trace after 112 days. With the standard treatment, in which captan was incorporated eight times in the program starting at the 7-day interval, leaf extracts showed mutagenic activity at 7, 14, 28 and 90 days. Captan residues at these intervals were 11.4, 5.0, 4.1 and 3.4 micrograms/cm2, respectively. Fruit sample extracts of the standard spray were mutagenic to the tester strains TA100 and TA98 both at mid-season and at harvest. Residues of captan on fruits declined from 10.4 micrograms/cm2 at mid-season to 1.1 micrograms/cm2 at harvest. No mutagenic activity was detected with extracts from fruit samples from the single captan application.     

Effects of salinity and ozone,individually and in combination,on the growth and ion contents of two chickpea (Cicer arietinum L.) varieties

The aim of this study was to determine the effects of ozone and salinity, singly and in combination, on the growth and ion contents of two chickpea (Cicer arietinum L.) varieties. Chickpea plants were grown in non-saline and saline conditions, with and without a repeated exposure to ozone. Salinity at a concentration of 30 mM NaCl caused a substantial reduction in plant height, number of leaves and the dry weights of the leaves, stems and roots. Biomass allocation to the leaves increased, predominantly at the expense of the roots. Ozone at a concentration of 85 nmol mol(-1) for 6 h per day for 25 days reduced plant height and dry weights but had no effect on leaf number. The results show substantial effects of salinity and ozone on chickpea growth and ion concentrations. When ozonated plants are grown in the presence of salinity, further reductions in growth occur.     

Earthworm ecotoxicological assessments of pesticides used to treat seeds under tropical conditions

Ecotoxicological laboratory tests (lower-tier tests) are fundamental tools for assessing the toxicity of pesticides to soil organisms. In this study, using these tests under tropical conditions, we quantified the impact of the insecticides imidacloprid, fipronil, and thiametoxam, and the fungicides captan and carboxin + thiram, all of which are used in the chemical treatment of crop seeds, on the survival, reproduction, and behavior of Eisenia andrei (Oligochaeta). With the exception of imidacloprid, none of the pesticides tested caused mortality in E. andrei in artificial soils. The LC₅₀ of imidacloprid was estimated as 25.53 mg active ingredient kg^?1 of dry soil. Earthworm reproduction rates were reduced by imidacloprid (EC₅₀ = 4.07 mg kg^?1), fipronil (EC₂₀ = 23.16 mg kg^?1), carboxin + thiram (EC₅₀ = 56.38 mg kg^?1), captan (EC₅₀ = 334.84 mg kg^?1), and thiametoxam (EC₅₀ = 791.99 mg kg^?1). Avoidance behavior was observed in the presence of imidacloprid (AC₅₀ = 0.11 mg kg^?1), captan (AC₅₀ = 33.54 mg kg^?1), carboxin + thiram (AC₅₀ = 60.32 mg kg^?1), and thiametoxam (AC₅₀ = >20 mg kg^?1). Earthworms showed a preference for soils with the insecticide fipronil. Imidacloprid was the most toxic of the substances tested for E. andrei. The avoidance test was the most sensitive test for most pesticides studied, but results varied between pesticides. These results offer new insights on the toxicity of pesticides used to treat seeds in tropical regions. However, they should be complemented with higher-tier tests in order to reduce the uncertainties in risk assessment.     

Potential of recycling gamma-irradiated sewage sludge for use as a fertilizer: a study on chickpea (Cicer arietinum)

The effects of gamma-irradiated sludge on the growth and yield of chickpea (Cicer arietinum) in pot cultures have been studied. Compared to plants grown only in soil, root length, fresh weight and dry weight of plants grown in soil supplemented with unirradiated sludge were found to be significantly reduced. This inhibition in growth was found to be nullified when plants were grown in soil supplemented with gamma-irradiated sludge, suggesting that gamma radiation induced inactivation of toxic substance(s) in sludge. The protein content of plants grown in soil supplemented with irradiated sludge was also found to be significantly increased compared to those grown with unirradiated or no sludge, after 45 days. There was no significant effect of gamma irradiated sludge on shoot length, total soluble sugars, starch content and yield of chickpea plants. The results obtained suggest that the sludge tested, and obtained from the digester of a conventional domestic sewage treatment plant, is inhibitory to several growth parameters. Gamma irradiation of sewage resulted in removal of this inhibition. This suggests a possibility of beneficial and safe recycling of gamma-irradiated sludge for agricultural uses.     

Evaluation of fluoranthene phytotoxicity in pea plants by Hill reaction and chlorophyll fluorescence

The effect of both increased concentrations (0.01 and 1 mg l(-1)) of fluoranthene (FLT) and the duration of exposure (18 and 25 days) on the growth and photosynthetic processes in pea plants (Pisum sativum L., cv. Garde) was investigated. FLT concentration in roots and shoot of pea plants was also determined. The obtained results demonstrated that the higher concentration of FLT (1 mg l(-1)) significantly inhibited the growth of the pea plants after 25 days of the application, also affected the content of photosynthetic pigments (chlorophyll a, b and carotenoids), and the primary photochemical processes of photosynthesis. In chlorophyll fluorescence parameters, the significant increase of F(0) values and the decrease of F(V)/F(M) and Phi(II) values was recorded. The Hill reaction of isolated chloroplasts of pea plants was significantly inhibited after 25 days by presence of FLT (0.01 and 1 mg l(-1)) in nutrient solution, while after 18 days no significant response of Hill reaction activity was recorded. The fluoranthene content in roots and shoot of pea plants increased with increasing FLT concentration in the environment and the substantial accumulation of FLT was observed in the roots.     

Effects of DDT on the growth of crop plants

The effects of DDT on the germination and growth of plants were studied using many crop species. Of the species tested, oil-rich seeds of plants, such as peanut (Arachis hypogaea) and mustard (Brassica juncea), were more prone to DDT induced inhibition of germination and subsequent plant growth than cereals, pulses and fibre crops, like rice (Oryza sativa), barley (Hordeum vulgare), mung bean Vigna radiata), pigeon pea (Cajanus cajan) and cotton (Gossypium hirsutum). Studies with (14)C labelled DDT showed that insecticide uptake by seeds was directly proportional to seed size. However, there was no direct relationship between DDT uptake by the seeds and its subsequent translocation to the growing regions or the degree of growth inhibition. Data suggest that oil content of the seeds per se has a bearing on the susceptibility or tolerance of a plant to DDT. It is suggested that lipids of the plant cell solubilize and disperse DDT in the cytoplasm, which, in turn, affects normal metabolism within the cell.     

Effect of cadmium on germination, amylases and rate of respiration of germinating pea seeds

Growth of embryonic axis of germinating pea seeds (Pisum sativum cv. Bonneville) was significantly inhibited by as low as 0.25 mM cadmium and the elongation of the radicle was affected more severely than that of the plumule. Total amylolytic activity, as well as activities of alpha- and beta-amylases, diminished progressively with increasing concentrations of the metal in the media. The deleterious effect on alpha-amylase persisted throughout, whereas beta-amylase activity recovered with time. The rate of respiration of seeds, measured by oxygen uptake, was markedly impeded by cadmium and the phase of rapid and linear development of respiratory activity (after 3 days of imbibition) was almost completely, suppressed in the presence of higher concentrations (1 mM) of the metal. The observed impact of cadmium on starch mobilization and on respiratory activity are discussed in relation to its adverse effects on seed germination.     

The effects of zinc on cell viability and on mitochondrial structure in contrasting cultivars of Festuca rubra L. - a rapid test for zinc tolerance

A 20-min exposure to 5.0 microg Zn cm(-3) reduced the percentage of viable root meristematic cells in three cultivars of Festuca rubra L.: Merlin (Zn-tolerant), Hawk (salt-tolerant but with a degree of Zn tolerance) and S59 (Zn-sensitive). The Zn-induced cell mortality in S59 was approximately twice that of the tolerant cultivars. The mean area of mitochondrial profiles in root meristematic cells of Zn-untreated roots was similar in S59 and Merlin but that of Hawk was smaller. A 4-day exposure to 0.2 microg Zn cm(-3) resulted in mitochondrial swelling in the Zn-sensitive cultivar; there was a 25% increase in the mean area of mitochondrial profiles in this cultivar, but no significant increase occurred in Hawk or Merlin. Zn treatment caused a collapse of the cristae and a localized condensation of the mitochondrial matrix in S59, but not in Hawk or Merlin. The marked increase in cell mortality after only a 20-min Zn exposure and the relative simplicity of the technique, indicates that this procedure could be used as a rapid and independent measure of Zn tolerance.     

Effects of DDT ground-spraying against tsetse flies on lizards in NW Zimbabwe

The impact of DDT ground-spraying against tsetse flies on lizards was investigated in NW Zimbabwe. Nineteen species were recorded, 17 in mopane woodland and 11 on gritstone outcrops: Mabuya striata wahlbergii dominated trees and Mabuya quinquetaeniata margaritifer rocks. Mean frequency of M. s. wahlbergii declined significantly from 76% of lizards at untreated sites (n = 8), through 72% after three annual treatments (n = 4), to 48% after 4-6 treatments (n = 6). Sighting rates and proportion of trees occupied were also significantly lower at treated than untreated sites. Numbers on trunks (99% > 15 cm diameter) above 3 m increased significantly with years of treatment relative to those in the spray target area below 3 m. Total DDT loads rose significantly with number of annual treatments and were up to 263 microg g(-1) lipid (7 microg g(-1) wet body weight) after 3-6 years. The percentage of unaltered DDT increased with load, which was proportionately higher in thin than in fat lizards. The geometric mean total DDT level in M. s. wahlbergii was significantly higher than in outcrop species, and from treated woodland was elevated 21 times above that in lizards from treated outcrops. Frequency and sighting rates of Lygodactylus chobiensis in woodland and immature Agama kirkii on outcrops were significantly higher in treated than in untreated areas.