Determination of 17 Organophosphate Pesticide Residues in Mango by Modified QuEChERS Extraction Method Using GC-NPD/GC-MS and Hazard Index Estimation in Lucknow,India

A total of 162 samples of different varieties of mango: Deshehari, Langra, Safeda in three growing stages (Pre-mature, Unripe and Ripe) were collected from Lucknow, India, and analyzed for the presence of seventeen organophosphate pesticide residues. The QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method of extraction coupled with gas chromatography was validated for pesticides and qualitatively confirmed by gas chromatography- mass spectrometry. The method was validated with different concentrations of mixture of seventeen organophosphate pesticides (0.05, 0.10, 0.50 mg kg⁻¹) in mango. The average recovery varied from 70.20% to 95.25% with less than 10% relative standard deviation. The limit of quantification of different pesticides ranged from 0.007 to 0.033 mg kg⁻¹. Out of seventeen organophosphate pesticides only malathion and chlorpyriphos were detected. Approximately 20% of the mango samples have shown the presence of these two pesticides. The malathion residues ranged from ND-1.407 mg kg⁻¹ and chlorpyriphos ND-0.313 mg kg⁻¹ which is well below the maximum residues limit (PFA-1954). In three varieties of mango at different stages from unpeeled to peeled sample reduction of malathion and chlorpyriphos ranged from 35.48%–100% and 46.66%–100% respectively. The estimated daily intake of malathion ranged from 0.032 to 0.121 µg kg⁻¹ and chlorpyriphos ranged from zero to 0.022 µg kg⁻¹ body weight from three different stages of mango. The hazard indices ranged from 0.0015 to 0.0060 for malathion and zero to 0.0022 for chlorpyriphos. It is therefore indicated that seasonal consumption of these three varieties of mango may not pose any health hazards for the population of Lucknow, city, India because the hazard indices for malathion and chlorpyriphos residues were below to one.     

Toxicity of certain insecticides toSturmiopsis inferens,a larval parasite of sugarcane moth borers

Susceptibility of adults and puparia ofSturmiopsis inferens Tns. [Tachinidae, Diptera] to 9 commonly recommended insecticide sprays against sugarcane pests was determined. The chemicals tested as emulsifiable concentrates include lindane 0.1%, endosulfan 0.1%, monocrotophos 0.05%, quinalphos 0.05%, malathion 0.1%, dimethoate 0.1%, cypermethrin 0.01%, fenvalerate 0.01% and decamethrin 0.0014%. Lindane, malathion, dimethoate monocrotophos and quinalphos were highly toxic, while decamethrin had little harmful effect to the adults when exposed for 6 h to filter paper impregnated or sugarcane shoot bits sprayed with the chemicals. However, the insecticides had no harmful effect on the puparia and adult emergence was normal from the puparia sprayed with insecticides. In another study, susceptibility of adults to soil application of lindane EC, carbofuran G, chlorpyriphos G, Sevidol G and whorl application of lindane G, chlorpyriphos G and Sevidol G was tested in pot culture. Except for soil application of lindane EC, all other chemicals had no harmful effect to the adults in pot culture experiment. In a field trial, commonly recommended insecticides against shoot borer,Chilo infuscatellus Snell.viz., soil application of granules of lindane, carbofuran, chlorpyriphos and Sevidol and folia spray of endosulfan did not affect the parasite activity. Institute Publication No 1030.     

Synthesis of Extracellular Chitinase by Wild-Type B-10 and Mutant M-1 Strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, whereas M-1 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused oversynthesis of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the synthesis of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).     

Evolutionary Diversification Indicated by Compensatory Base Changes in ITS2 Secondary Structures in a Complex Fungal Species, <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The rRNA cistron (18S–ITS1–5.8S–ITS2–28S) is used widely for phylogenetic analyses. Recent studies show that compensatory base changes (CBC) in the secondary structure of ITS2 correlate with genetic incompatibility between organisms. Rhizoctonia solani consists of genetically incompatible strain groups (anastomosis groups, AG) distinguished by lack of anastomosis between hyphae of strains. Phylogenetic analysis of internal transcribed spacer (ITS) sequences shows a strong correlation with AG determination. In this study, ITS sequences were reannotated according to the flanking 5.8S and 28S regions which interact during ribogenesis. One or two CBCs were detected between the ITS2 secondary structure of AG-3 potato strains as compared to AG-3 tobacco strains, and between these two strains and all other AGs. When a binucleate Rhizoctonia species related to Ceratobasidiaceae was compared to the AGs of R. solani, which were multinucleate (3–21 nuclei per cell), 1–3 CBCs were detected. The CBCs in potato strains of AG-3 distinguish them from AG-3 tobacco strains and other AGs yielding further evidence that the potato strains of AG-3 originally described as R. solani are a species distinct from other AGs. The ITS1–5.8S–ITS2 sequences were analyzed by direct sequencing of PCR products from 497 strains of AG-3 isolated from potato. The same 10 and 4 positions in ITS1 and ITS2, respectively, contained variability in 425 strains (86%). Nine different unambiguous ITS sequences (haplotypes) could be detected in a single strain by sequencing cloned PCR products indicating that concerted evolution had not homogenized the rRNA cistrons in many AG-3 strains. Importantly, the sequence variability did not affect the secondary structure of ITS2 and CBCs in AG-3. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Resistance to chlorpyriphos in the predatory mite Kampimodromus aberrans

The predatory mite Kampimodromus aberrans (Oudemans) is a key biocontrol agent in vineyards in Italy and Southern Europe. Its susceptibility to common pesticides (e.g., organophosphates) has been considered an important factor in preventing successful biocontrol of phytophagous mites. Nevertheless, populations of K. aberrans apparently resistant to organophosphates (OPs) have been reported to occur in Northern Italian vineyards. The resistance of K. aberrans to fungicides (e.g., mancozeb) has been demonstrated in the laboratory in France, but little is known about the toxicity of insecticides towards K. aberrans. Of these pesticides, the OP chlorpyriphos is extensively used in viticulture to control lepidopterans and homopterans. The present study investigated the dose–response effect of chlorpyriphos in four K. aberrans strains characterized by different levels of exposure to OP insecticides in the past: from never to frequently exposed. Resistance to chlorpyriphos is demonstrated for populations collected from vineyards and apple orchards. Resistance factors exceeded 145,000× for the three strains collected in vineyards and orchard. LC₅₀ values for resistant strains were 1.85–6.83 times higher than the recommended field dose of chlorpyriphos for vineyards and orchards (525 mg a.i./l).     

The absence of a correlation between plasmids and luminescence in marine luminous bacteria

Members of four species of marine luminous bacteria were examined for the presence of plasmids using gel electrophoresis of purified alkaline extracts. One to four plasmids, with molecular weights ranging from 5 to 120 megadaltons, were found to occur in 43% of the 58 bioluminescent strains examined. There was, thus, no correlation between the presence and absence of plasmids and luminescence, nor was there any single size of plasmid common to the different bacterial species. Spontaneous dark (dim) mutants were selected from five strains ofVibrio (Beneckea) harveyi; in no case was there any difference in the plasmid content between the bright parent strain and the dim isolate.     

Experimental biofilm and its application in malathion degradation

Mixed bacterial culture consisting of three different strains ofMicrococcus sp. (AG 36, AG 94 and AG 98) and two strains ofPseudomonas sp. (AG 7 and AG 52) and its individual components was passed through a sand column and 25.5–92% of cell dry mass was found to be retained (adsorbed) on it. Incubation of sand soaked in mineral medium containing glucose as a sole carbon source resulted in formation of a biofilm with 1.2–2.5-fold increase in biomass. A 61% degradation of malathion by the mixed culture biofilm could be achieved in 4 d.     

Characterization of new plasmids from methylotrophic bacteria

Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotrophMethylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genusMethylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and theE. coli plasmid pK19 Km^r, which were checked for conjugative transfer fromE. coli into the methylotrophic host.     

Biodegradation of malathion by <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain KB2 and <Emphasis Type="Italic">Bacillus cereus</Emphasis> strain PU

We report here the degradation of a pesticide, malathion, by Brevibacillus sp. strain KB2 and Bacillus cereus strain PU, isolated from soil samples collected from malathion contaminated field and an army firing range respectively. Both the strains were cultured in the presence of malathion under aerobic and energy-limiting conditions. Both strains grew well in the medium having malathion concentration up to 0.15%. Reverse phase HPLC–UV analysis indicated that Strain KB2 was able to degrade 72.20% of malaoxon (an analogue of malathion) and 36.22% of malathion, while strain PU degraded 87.40% of malaoxon and 49.31% of malathion, after 7 days of incubation. The metabolites mal-monocarboxylic acid and mal-dicarboxylic acid were identified by Gas chromatography/mass spectrometry. The factors affecting biodegradation efficiency were investigated and effect of malathion concentration on degradation rate was also determined. The strain was analyzed for carboxylesterase activity and maximum activity 210 ± 2.5 U ml⁻¹ and 270 U ± 2.7 ml⁻¹ was observed for strains KB2 and PU, respectively. Cloning and sequencing of putative malathion degrading carboxylesterase gene was done using primers based PCR approach.     

Isolation and Characterization of Bile Acid 7-Dehydroxylating Bacteria from Human Feces

Methods for isolation of fecal 7α-dehydroxylating bacteria are presented. A total of 219 strains were isolated from feces of healthy humans, and their ability to 7-dehydroxylate cholic, chenodeoxycholic, and ursodeoxycholic acids were examined. Of all the isolates, 14 strains were found to be capable of eliminating the hydroxy group at C-7α and/or C-7β. All the isolates were strictly anaerobic, Gram-positive rods. Thirteen isolates were non-sporeforming bacteria showing certain saccharolytic properties with the production of acid and gas from dextrose, and were catalase-positive but indole-, lecithinase-, urease- and oxidase-negative. Based on the data available at present, it was concluded that they could be regarded as members of the genus Eubacterium. One strain, however was identified as Clostridium sordellii. The isolated strains capable of 7α-dehydroxylating cholic acid and chenodeoxycholic acid were also able to oxidize the hydroxy group at C-7α. Nine strains (10, 12, 36S, M-2, M-17, M-18, Y-98, Y-1112, and Y-1113) of the 7α-dehydroxylating bacteria were confirmed to have 7β-dehydroxylation ability, but five strains (O-51, O-52, O-71, O-72, and Y-67) could not transform ursodeoxycholic acid to lithocholic acid.     

Involvement of a Polyethylene glycol (PEG)-oxidizing Enzyme in the Bacterial Metabolism of PEG

Eighty-seven strains of acetic acid bacteria were surveyed for plasmids by CsCl-ethidium bromide equilibrium centrifugation of cell lysates. Twenty-seven of the 33 strains of Acetobacter were found to harbor plasmid DNA and most strains contained multiple species of low-molecular-weight plasmids. On the other hand, plasmid DNAs were detected in 23 of the 36 strains of Gluconobacter and most of them had molecular weights of more than 5 megadaltons. Of the 18 strains newly isolated from a vinegar factory, some of which were examined taxonomically, 17 contained plasmid DNA. The molecular weights of plasmids detected in this study were in the range of about one to over 17 megadaltons. The plasmids with molecular weights of less than 5 megadaltons were characterized with restriction endonucleases. The physical maps of two plasmids designated as pMV1O1 and pMV102, which were found in the isolated strains, were constructed.     

Performance ofin vitro propagatedAlnus glutinosa (L.) Gaertn. clones inoculated withFrankiae

Summary ThreeAlnus glutinosa (L.) Gaertn. clones, obtained byin vitro propagation techniques, were inoculated with four strains ofFrankia. The ability of these clones to nodulate and fix nitrogen was previously reported; this study deals with the performance of 12 different combinations of pairs of symbionts.Shoot fresh weight, shoot height and collar diameter were measured 60 and 82 days after inoculation. Shoot fresh weight seems to be more sensitive and reliable than the other parameters. Nitrogenase activity, measured by the acetylene reduction assay, was assayed 78 days after inoculation and was consistent with the biomass measurements.Better growth was observed when type N strains were used. Significant growth differences were observed between clones AG-2 and AG-8 on the one hand and clone AG-4 on the other. Thus, the use of genetically defined host plants and microsymbionts permitted the demonstration of significant performance variation even among cloned plants from the same provenance (AG-4 and AG-8).The duration of the experiment influenced the results with differences becoming less significant with time. This might be caused by an external limiting factor such as the pot size, competition for light,etc. But it could also be indicative of differences in nodulation speed among the treatments.     

Extracellular chitinase production by wild-type B-10 and mutant M-1 strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, while M-10 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused overproduction of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the production of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).     

Susceptibility of malathion-resistant strains of Tribolium castaneum and T. confusum to the insect growth regulators methoprene and hydroprene

A comparison was made of the effects of methoprene (isopropyl 11-methoxy-3, 7, 11-trimethyl-2, 4-dodecadienoate) and hydroprene (ethyl 3, 7, 11-trimethyl dodeca-2, 4 dienoate) on the developmental survival of three malathion-resistant strains of Tribolium castaneum (Herbst) and one of T. confusum J. du Val, with a malathion-susceptible strain of each species. No cross-resistance to either IGR in any of the malathion-resistant strains was detected.

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Résumé On a fait une comparaison des effets de methoprene (isopropyl 11-methoxy-3, 7, 11-trimethyl-2, 4-dodecadienoate) et hydroprene (ethyl 3, 7, 11-trimethyldodeca-2, 4 dienoate) sur le développement de trois races de Tribolium castaneum résistantes au malathion, et une race de T. confusum résistante au malathion, avec une race de chaque espèce prédisposée au malathion. Aucune résistance croisée a l'un ou l'autre IGR ne fut découverte dans les races résistantes au malathion.

     

Transfer and stability of drug resistance plasmids inEscherichia coli K12

Mating experiments between pairs of strains ofEscherichia coli containing either the compatible plasmids TP120 (Inc N) and R1 (Inc FII) or the incompatible plasmids TP125 (Inc B) and TP113 (Inc B) were undertaken in mixed continuous-flow cultures and in dialysis sacs suspended in pond water. Plasmid transfer was readily demonstrated between strains carrying compatible plasmids TP120 and R1 in both continuous-flow culture and pond water. In mixed cultures of strains carrying plasmids TP125 and TP113, transfer was only observed in continuous-flow culture systems. Strains ofE. coli containing aggregates of plasmids TP120 and R1 were shown to be stable over 5 months continuous cultivation under carbon limited conditions at a growth rate of 0.1 hours^–1 in the presence of drugs which select for the maintenance of both plasmids. In the strains containing plasmid aggregates, a gene dosage effect was observed with respect to the levels of resistance to drugs whose resistance was encoded by both plasmids. Chemostat experiments showed that no cointegrate plasmids were found from the strains ofE. coli initially containing both plasmid TP120 and plasmid R1.     

The Cotransduction of pET System Plasmids by Mutants of T4 and RB43 Bacteriophages

The study of the cotransduction of the plasmid pairs pET-3a–pLysE and pET-3a–pLysS by the mutant phage T4alc7 showed that the antibiotic resistance markers of the plasmids were cotransduced with a high frequency. The analysis of the plasmid DNA of cotransductants and cotransformants showed that the mutant phage T4alc7 can be used for obtaining the monomeric and oligomeric forms of plasmids and for the cotransduction of two-plasmid overproduction systems into E. coli strains. The plaque mutants RB43-03 and RB43-13 derived from bacteriophage RB43 were found to be able to cotransduce the antibiotic resistance markers of pET-3a and pLysE plasmids.     

Plasmids from bacterial endosymbionts of hump-killer paramecia

Six isolates ofCaedibacter taeniospiralis, collected from four continents, were screened for plasmid DNA. Plasmid DNA species containing between 41.5 and 49.5 kilobase pairs (kb) were observed in all strains. Physical maps of plasmids were constructed by determining relative positions of the restriction endonuclease (BamHI,SalI,XhoI,SacI,PstI,AvaI, andEcoRI) recognition sequences in each plasmid. The physical map of the smallest plasmid (41.5 kb), pKAP30, is reflected in each of the plasmids isolated from the other strains ofC. taeniospiralis. Plasmid DNA from three of the isolates (strains 51 and 116 both from Indiana and strain 169 from Japan) each contain 43 kb, where 41.5 kb appear to be identical to pKAP30 (obtained from the Australian strain, A30). The extra 1.5 kb present in pKAP51, pKAP116, and pKAP169 is included as a single polynucleotide sequence. The 1.5-kb inclusion is located at apparently identical positions in pKAP116 and pKAP169 and at a totally different position in pKAP51. The two remaining plasmids, pKAP47 (from California strain 47) and pKAP298 (from Panama strain 298), both contain 49 kb to include a continuous 41.5-kb sequence that is apparently identical to pKAP30. The results indicate that the polynucleotide sequences of these plasmids are highly conserved and that the observed variations among them may be accounted for by transposable elements.     

Comparison of Low-Molecular-Weight Heat Stress Proteins Encoded on Plasmids in Different Strains of Streptococcus thermophilus

Streptococcus thermophilus is used extensively for industrial fermentation of dairy products. Some strains of S. thermophilus are known to carry plasmids, and many of these plasmids are suspected of encoding low-molecular-weight heat stress proteins (Hsps) that may aid in survival under stressful conditions. In order to confirm the presence and examine the similarity of these low-molecular-weight Hsps, genes were identified and sequenced encoding Hsps on plasmids pER16 (4.5 kb), pER35 (10 kb), and pER36 (3.7 kb) from three different strains of S. thermophilus. The plasmid replication proteins were also sequenced to examine their relatedness. Amino acid sequence comparisons of the Hsps and of the replication proteins revealed a high degree of identity suggesting a common origin. Heat stress proteins enhance the viability of bacteria in extreme environments, and the presence of an Hsp encoded on a plasmid may enhance survival of S. thermophilus under harsh production conditions. Received: 8 February 2000 / Accepted: 8 March 2000     

Strong and weak immune responses across the same major histocompatibility barrier in rats

Inbred rat strains, Fischer 344 (F-344) and Lewis (LEW), share the serologicalAg-Bl allele and react very weakly in mixed lymphocyte culture (MLC). Despite this apparent identity atAg-B, these strains differ markedly in their immune responses to anAg-B disparate third strain Marshall 520 (M-520) (Ag-B6). F-344 recipients allowed M-520 heart grafts an extended survival, whereas LEW recipients rejected them rapidly. F-344 and M-520 showed a weak response in MLC in contrast to a strong response for LEW and M-520. F-344 produced antisera in response to injection of M-520 cells that had a relatively high antibody titer but low cytotoxic activity. F-344 responded to another strain, Buffalo (BUF) (alsoAg-B6), in a similar fashion. F-344 apparently can produce a strong allogeneic response, as it was able to rapidly reject heart grafts from (LEW x Brown-Norway) F₁ donors (LBN) (Ag-B 1/3). The low response of F-344 to M-520 probably was not due to shared antigens between the two strains because M-520 heart grafts underwent rapid rejection in LEW hosts highly tolerant to F-344. To explain the contrasting response of F-344 and LEW to theAg-B6 disparity, we propose that it is controlled by an immune-response gene(s); that F-344 has a low-responding allele and LEW has a high-responding allele. The data do not reveal a location for this proposed gene. The high-responding allele appears to be dominant, as M-520 hearts were rejected rapidly by (F-344 x LEW) F₁ recipients.     

Insecticide resistance and malathion carboxylesterase in the sheep blowfly,Lucilia cuprina

Resistance to the organophosphorus insecticide malathion in genetically related strains of the Australian sheep blowflyLucilia curprina was examined. Separate lines of blowflies were established by homozygosis of the fourth chromosome of the parental RM strain. Both the RM and the derived resistant (der-R) strains are approximately 100 times more resistant to malathion than the related susceptible der-S strain, resistance being correlated with a 45- to 50-fold increase in a malathion carboxylesterase (MCE) activity. MCE has a pH optimum ranging between 6.6 and 8.0 and is strongly inhibited by the carboxylesterase inhibitors triphenyl phosphate, paraoxon, and diiospropylfluorophosphate. Subcellular fractionation revealed that MCE was localized predominantly to the cytosol and mitochondria in both resistant and susceptible blowflies. A single MCE was purified to homogeneity from RM blowflies. It has a pI of 5.5, is a monomer of 60.5 kDa, and hydrolyzes malathion with aV _max of 755 nmol/min/mg protein and aK _m of 11.0 µM. L. cuprina have thus evolved a remarkable MCE which is faster and more efficient at hydrolyzing a specific insecticide than any other insect esterase yet described.