Low Residual Toxicity Research Articles

Antifungal activity and preliminary mechanism of pristimerin against Sclerotinia sclerotiorum

Sclerotinia sclerotiorum (Lib.) de Bary is a crop-threatening fungus. Natural products are ideal antimicrobial agents to combat pathogens. They are considered a promising alternative to traditional synthetic fungicides due to low residual toxicity and possibly different modes of action on organisms. This work aims to develop a leading compound from natural products. Here, the antifungal activity of ten pentacyclic triterpenoids, including celastrol, pristimerin, asiatic acid, maslinic acid, ursolic acid, oleanic acid, quillaic acid, glycyrrhetinic acid, madecassic acid and corosolic acid were evaluated. Pristimerin exhibited an excellent activity against Sclerotinia sclerotiorum (S. sclerotiorum) with an EC50 value of 1.36 µg/mL. Subsequently, the mode of action of pristimerin on S. sclerotiorum was further characterized by RNA-Seq-Based transcriptome profile. Pristimerin mainly inhibited oxidative phosphorylation, citrate cycle (TCA cycle) and blocked energy metabolism, and it also promoted the accumulation of ROS and disrupted the balance of the active oxygen metabolism system by affecting the activities of peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) in the mycelial cells, and ultimately led to cell death. And beyond that, mitochondrial damage and changes in mycelial cells were also observed by transmission electron microscope (TEM) and scanning electron microscope (SEM). In general, these findings showed a molecular level perspective on the antifungal mechanism of pristimerin against S. sclerotiorum, and it could be used as a potential fungicide against S. sclerotiorum.

Toxicology of diatomaceous earth, phyto oils and their admixed emulsions against adults of Tribolium castaneum (Herbst)

Tribolium castaneum (Herbst), one of the invasive stored pest, is resistant to the most of synthetic insecticides used against this it. Moreover, synthetic insecticides are a major threat to human health, the environment, and the ecosystem. The development of some smart tools is needed to minimize the use of hazardous chemicals. As an alternative, nano-insecticides are on the horizon. Emulsions are expressed as sustain release of insecticidal components to achieve maximum efficacy and low residual toxicity. In this study, some essential oils (Cymbopogon citratus (DC.) Stapf, Ocimum basilicum L., Curcuma longa L., and Trachyspermum ammi L.), diatomaceous earth (DE), and their nano-emulsions are evaluated against T. castaneum. Essential oils and DE were tested at four (60 ppm, 30 ppm, 15 ppm, 7.5 ppm) different concentrations with and without emulsions, and data was recorded after 6, 12, 24, 48, and 72 h of exposure respectively. The mortality observed in essential oils of C. citratus, O. basilicum, and C. longa without emulsion after 72 h of treatment at the highest concentration was 98%, 95%, and 85%, respectively. While, at the highest concentration the lowest mortalities were observed with DE and T. ammi i.e. 65%. Insecticidal activity of emulsion essential oils increased to 100%, 98%, 90%, and 68.3% for C. longa, C. citratus, O. basilicum, and T. ammi, respectively. The results support that these admixed emulsions could be used as an alternative to synthetic insecticides in conventional formulations.

Red mud modified sludge biochar for the activation of peroxymonosulfate: Singlet oxygen dominated mechanism and toxicity prediction

In this paper, red mud-sewage sludge derived biochar (RSDBC) was synthesized and employed as the heterogenous activator of peroxymonosulfate (PMS) for sulfamethoxazole (SMX) degradation. With the incorporation of red mud, 82.5% degradation of SMX was achieved by RSDBC/PMS system in a process dominated by 1O2, which was attributed to the participation of oxygen vacancy, ketone groups and graphitic carbon. On the other hand, in the absence of red mud, OH and SO4•− were dominantly accounted for SMX degradation in sewage sludge derived biochar (SDBC)/PMS system. In this case heterogeneous Fe species, ketone groups and graphitic carbon were responsible for PMS activation. Due to the different Reactive Oxygen Species (ROS), effects of reaction conditions including initial pH, common anions and natural organic matter (NOM) were not in full accord. Besides, Fe leaching from RSDBC (0.67 ppm) was much lower than that of SDBC (3.07 ppm), leading to a better reuse ability for RSDBC. Less degradation intermediates were disclosed in RSDBC/PMS system, along with lower residual toxicity. In addition, eco-toxicity of all the intermediates was predicted by ECOSAR program for the further understanding of the detoxification of SMX. Advantages of RSDBC/PMS system as disclosed in this paper further suggest its potential full-scale application of environmental remediation.

Broad-spectrum antifungal activity of dichloromethane extract of Waltheria indica stems and isolated compounds

The secondary metabolites produced by plants are important sources of fungicides with intriguing bioactivities and environmentally benign attributes (eg. low residual toxicity to mammals and readily biodegradable in soil). In this study, the dichloromethane extract of stems of Waltheria indica L. was found to exhibit broad-spectrum antifungal activity toward eight phytopathogenic fungi, particularly against carbendazim-resistant Sclerotinia sclerotiorum with inhibition rate of 100% at 500 μg/mL. Two quinoline alkaloids, antidesmone and waltherione C, were isolated and identified as fungicidal compounds by bioassay-guided isolation and spectroscopic analyses. Antidesmone displayed superior antifungal activities against eight phytopathogenic fungi to commercial botanical fungicides (osthole, carvacrol, and eugenol). Moreover, antidesmone was found to have significant negative cross-resistance with carbendazim to Sclerotinia sclerotiorum for the first time and exhibited much better antifungal activity (EC50 = 0.60 μg/mL) against carbendazim-resistant strains than carbendazim-sensitive strain (EC50 = 9.61 μg/mL) of Sclerotinia sclerotiorum, which was further supported by the molecular modelling result. In addition, the dichloromethane extract of the Waltheria indica stems could effectively control the development of eleven species of phytopathogenic fungi in vivo. These results suggest that Waltheria indica dichloromethane extract and antidesmone can be developed as promising broad-spectrum botanical fungicides.

Synthetic lipids as a biocide candidate for disinfection of ballast water

The objective of this study is to propose the use of specific synthetic lipid as an active substance (biocide) in the control of harmful aquatic microorganisms, such as pathogens and non-indigenous species, transported in ships' ballast water. The biocide candidate, without metal or halogen components, was produced from a sub-product of the edible oil industry, the lecithin. Laboratory assays were conducted with phytoplankton, zooplankton, and marine bacteria to evaluate the efficiency of the biocide. The study also considers specific biocide's characteristics related to environmental risks, such as chemical composition, persistence, bioaccumulation, and toxicity. Results showed that, in the first 24 h of treatment, the biocide effectively reduced the concentration of the planktonic micro-organisms to very low levels. Additionally, a preliminary risk evaluation pointed that biocide candidate has a low residual toxicity, also a low potential for persistence and bioaccumulation in the environment.

Efficient degradation of triclosan by an endophytic fungus Penicillium oxalicum B4

Triclosan (TCS), a widely used antimicrobial and preservative agent, is an emerging contaminant in aqueous and soil environment. Microbial degradation of TCS has not been reported frequently because of its inhibition of microbe growth. To explore the new microbial resources for TCS biodegradation, fungal endophytes were isolated and screened for the degradation potential. The endophytic strain B4 isolated from Artemisia annua L. showed higher degradation efficiency and was identified as Penicillium oxalicum based on its morphology and ITS sequences of ribosomal DNA. In both medium and synthetic wastewater, TCS (5mg/L) was almost completely degraded within 2h by the strain B4. The high capacity of TCS uptake (127.60 ± 8.57mg/g dry weight, DW) of fungal mycelium was observed during the first 10min after TCS addition. B4 rapidly reduced initial content (5.00mg/L) of TCS to 0.41mg/L in medium in 10min. Then, the accumulation of TCS in mycelium was degraded from 0.45 to 0.05mg/g DW after 1-h treatment. The degradation metabolites including 2-chlorohydroquinone, 2, 4-dichloropheno, and hydroquinone were found to be restrained in mycelia. The end products of the biodegradation in medium showed no toxicity to Escherichia coli. The new characteristics of high adsorption, fast degradation, and low residual toxicity highlight the potential of endophytic P. oxalicum B4 in TCS bioremediation.

Toxicities of 26 pesticides against 10 biological control species

To determine selective effectiveness for specific pesticides on biological control species we evaluated the contact toxicity of 16 insecticides, 2 acaricides, 3 fungicides, and 5 biopesticides. Targeted species included 3 predatory mites (Phytoseiulus persimilis Athias-Henriot, Amblyseius swirskii Athias-Henriot, and Neoseiulus californicus McGregor), 5 hymenopteran parasitoids (Diglyphus isaea Walker, Aphidius colemani Viereck, Encarsia formosa Gahan, Eretmocerus eremicus Rose and Zolnerowich and E. mundus Mercet), and 2 hemipteran predators (Orius laevigatus Fieber and Nesidiocoris tenuis Reuter) in laboratory condition. In addition, residual toxicity was evaluated on P. persimilis, E. formosa, and O. laevigatus. For contact toxicity, five insecticides (spinetoram, spinosad, lepimectin, chlorfenapyr, and dinotefuran+spinetoram) showed high toxicity to predatory mites. Most pesticides tested were highly toxic to all hymenopteran species except for D. isaea which showed low susceptibility to 11 pesticides. Bistrifluron+flubendiamide and B. valismortis were less toxic to A. colemani, and only B. valismortis was safe to both E. mundus and E. eremicus. Insect growth regulators (methoxyfenozide and bistrifluron), chlorantraniliprole, and bistrifuron+flubendiamide were not toxic to hemipteran predators. Fungicides and biopesticides were safe to hemipteran predators except for two biopesticides (B. subtilis and P. fluorescens). Most pesticides had low residual toxicity to P. persimilis, with the exception of chlorfenapyr whose toxicity persisted over 7days. One insecticide (cyantraniliprole), 2 acaricides (spiromesifen and fenpyroximate), 1 fungicide (metrafenone), and 4 biopesticides (B. subtilis, P. polymyxa, P. fluorescens, and B. valismortis) showed a much lower residual toxicity to E. formosa. Eight insecticides, 2 acaricides, 3 fungicides, and 5 biopesticides showed low residual toxicity to O. laevigatus.

사과과수원의 노린재류에 대한 농약의 생물활성 평가

노린재류는 전작물과 과수를 포함하여 다양한 작물에 피해를 준다. 특히 사과원에서는 썩덩나무노린재와 갈색날개노린재의 피해가 심하다. 사과과실을 선호하는 2종 노린재류(썩덩나무노린재와 갈색날개노린재)에 대하여 적용약제로 등록된 7종(dinotefuran WP, etofenprox WP, chlorpyrifos WP, cabaryl WP, chlothianidin SC, flonicamid WG, and bifenthrin WG)으로 접촉독성과 잔효활성에 대해 수행하였다. 접촉독성 실험에서 디노테퓨란은 갈색날개노린재 수컷에 처리후 48시간에서 96.7%, 썩덩나무노린재 수컷에 처리후 48시간에서 74.5%의 유의성 있는 접촉독성을 보였지만 다른 약제들은 효과가 낮았다. 이들 노린재류에 대한 접촉독성은 암컷보다 수컷에서 더 높았다. 실내조건에서 플로니카미드를 제외한 모든 약제들은 갈색날개노린재 암수모두에 잔효활성의 효과가 있었고, 반면에 클로로피리포스와 비펜트린이 썩덩나무노린재 암수에 대해서 잔효효과가 높았다. 야외에서 비펜트린은 처리후 5일차까지 갈색날개노린재에 대해 높은 잔효효과를 보였고, 썩덩나무노린재에 대해서는 다소 떨어지는 잔효효과를 보였다. 하지만 클로피리포스는 실내 잔효활성 실험에서 높은 잔효효과를 보였으나 야외에서는 두종 노린재에 대하여 낮은 잔효효과를 보였다. Stink bugs do damage on various crops including upland crops and tree fruits. Especially, yellow-brown stink bug (Halyomorpha halys (<TEX>$St{\aa}l$</TEX>)) and brown-winged green (Plautia stali) are severely damaged on apple orchard. Using seven insecticides - dinotefuran WP, etofenprox WP, chlorpyrifos WP, cabaryl WP, chlothianidin SC, flonicamid WG, and bifenthrin WG - registered on apple, contact and residual toxicities were tested on both male and female of P. stali and H. halys that preferred apple fruit. Contact toxicity of dinotefuran WP was excellent on male P. stali 48 hours after treatment (HAT) with 96.7% and significant on male Halyomorpha halys 48 HAT with 74.5% but the others had low effect. Contact toxicity on these stink bugs were higher in male than female. All insecticides except flonicamid, residual effects were all effective on both male and female of P. stali, while chlorpyrifos and bifenthrin were showed higher residual toxicity on both male and female of H. halys in laboratory condition. Two insecticides, chlorpyrifos and bifenthrin, were selected for the field test. Bifenthrin have a high residual effect on P. stali until 5 days after treatment, but have a low residual toxicity on H. halys in the field test. Chlorpyrifos showed higher residual toxicity in the laboratory, however, showed low residual efficacy on two species stink bug onto the field.

Adaptation of biomixtures for carbofuran degradation in on-farm biopurification systems in tropical regions.

A biomixture constitutes the active core of the on-farm biopurification systems, employed for the detoxification of pesticide-containing wastewaters. As biomixtures should be prepared considering the available local materials, the present work aimed to evaluate the performance of ten different biomixtures elaborated with by-products from local farming, in the degradation of the insecticide/nematicide carbofuran (CFN), in order to identify suitable autochthonous biomixtures to be used in the tropics. Five different lignocellulosic materials mixed with either compost or peat and soil were employed in the preparation of the biomixtures. The comprehensive evaluation of the biomixtures included removal of the parent compound, formation of transformation products, mineralization of radiolabeled CFN, and determination of the residual toxicity of the process. Detoxification capacity of the matrices was high, and compost-based biomixtures showed better performance than peat-based biomixtures. CFN removal over 98.5% was achieved within 16 days (eight out of ten biomixtures), with half-lives below 5 days in most of the cases. 3-Hydroxycarbofuran and 3-ketocarbofuran were found as transformation products at very low concentrations suggesting their further degradation. Mineralization of CFN was also achieved after 64 days (2.9 to 15.1%); several biomixtures presented higher mineralization than the soil itself. Acute toxicity determinations with Daphnia magna revealed a marked detoxification in the matrices at the end of the process; low residual toxicity was observed only in two of the peat-based biomixtures. Overall best efficiency was achieved with the biomixture composed of coconut fiber-compost-soil; however, results suggest that in the case of unavailability of coconut fiber, other biomixtures may be employed with similar performance.

The LTR72 mutant of heat-labile enterotoxin of Escherichia coli enhances the ability of peptide antigens to elicit CD4(+) T cells and secrete gamma interferon after coapplication onto bare skin.

Application of antigens with an adjuvant onto bare skin is a needle-free and pain-free immunization procedure that delivers antigens to the immunocompetent cells of the epidermis. We tested here the immunogenicity and adjuvanticity of two mutants of heat-labile enterotoxin (LT) of Escherichia coli, LTK63 and LTR72. Both mutants were shown to be immunogenic, inducing serum and mucosal antibody responses. The application of LTK63 and LTR72 to bare skin induced significant protection against intraperitoneal challenge with a lethal dose of LT. In addition, both LT mutants enhanced the capacity of peptides TT:830-843 and HA:307-319 (representing T-helper epitopes from tetanus toxin and influenza virus hemagglutinin, respectively) to elicit antigen-specific CD4(+) T cells after coapplication onto bare skin. However, only mutant LTR72 was capable of stimulating the secretion of high levels of gamma interferon. These findings demonstrate that successful skin immunization protocols require the selection of the right adjuvant in order to induce the appropriate type of antigen-specific immune responses in a selective and reliable way. Moreover, the use of adjuvants such the LTK63 and LTR72 mutants, with no or low residual toxicity, holds a lot of promise for the future application of vaccines to the bare skin of humans.

Surface-immobilized dextran limits cell adhesion and spreading

Dextran has recently been investigated as an alternative to polyethylene glycol (PEG) for low protein-binding, cell-resistant coatings on biomaterial surfaces. Although anti-fouling properties of surface-grafted dextran and PEG are quite similar, the multivalent properties of dextran are advantageous when high-density surface immobilization of biologically active molecules to low protein-binding surface coatings is desired. The preferred methods of dextran immobilization for biomaterial applications should be simple with minimal toxicity. In this report, a method is described for covalent immobilization of dextran to material surfaces which involves low residual toxicity reagents in mild aqueous reaction conditions. 70 kDa MW dextran was immobilized on glass and polyethylene terephthalate (PET) surfaces. 3T3 fibroblast cell adhesion was compared on untreated, aminated, and dextran-coated materials. Dextran coatings effectively limited cell adhesion and spreading on glass and PET surfaces in the presence of serum-borne cell adhesion proteins. With dextran-based surface coatings, it will be possible to develop well-defined surface modifications that promote specific cell interactions and perhaps better performance in long-term biomaterial implants.

Toxicity of the Ecdysone Agonist Tebufenozide to Codling Moth (Lepidoptera: Tortricidae)

The ecdysone agonist tebufenozide (RH-5992) has ovicidal, larvicidal but no adulticidal activity against codling moth, Cydia pomonella (L.). The toxicity to eggs varied with the application and with the substrate on which eggs were laid. On apple leaves treated in a spray tower, the ovicidal activity of tebufenozide was 30 times greater when eggs where laid on top of the residue than with topical application of eggs after they were laid (LC 50 s of 4.35 and 123.8 ppm, respectively). Eggs laid on apples treated in a spray tower were not affected by tebufenozide residue. However, tebufenozide had low residual toxicity to eggs (LC 50 = 117.47 ppm, flat dose-response line) when fruit was dipped instead of sprayed, suggesting some residual control on the fruit surface with airblast applications in the field. The LC 50 s for neonate larvae feeding on treated apples and on treated artificial diet were 16.08 and 0.22 ppm, respectively. In tests with artificial diet, susceptibility to tebufenozide decreased with larval age. Bioassays with artificial diet established reference lines for tebufenozide susceptibility in different instars. Contact exposure of neonates to tebufenozide residue on treated leaves or on a plastic surface caused mortality in larvae. Larval mortality increased with the duration of contact exposure. The effect of contact exposure on larval mortality was greater on treated apple leaves than on a treated plastic surface. Tebufenozide had no direct toxicity to adults (both sexes) and their longevity was not affected. Because of its ovicidal and larvicidal activity there is more flexibility in terms of spray timing with tebufenozide than with other insect growth regulators (e.g., diflubenzuron, fenoxycarb) which are primarily effective against the egg stage.

Evaluation of Toxicity of Different Pesticides to the Green lacewing, Mallada boninensis (Chrysopidae:Neuroptera)

The green lacewing, Mallada boninensis (Okamoto) is an important predator or sucking insects like mealybugs, syrphids and psyllids. A total or 34 commercial pesticides (18 insecticides, 16 acaricides/fungicides) on the larvae and adults of M. boninensis was studied for their initial toxicity at field recommended concentrations. Larvae were less susceptible than the adults. Fenvalerate, endosulfan, methyl demeton and synthetic pyrethrolds were less toxic to tbe larvae. None or the insecticides tested was safe to the adults or M. boninensis . But the fungicides proved harmless to both the larvae and adults. The insecticides which had shown high initial toxicity were further tested for their residual toxicity to both the stages. Insecticides like dichlorvos, methyl demeton, endosulfan and synthetic pyrethrolds had low residual toxicity and hence could be incorporated into the integrated control programme.

Volume 176, No. 1

1. The abilities of botulinum neurotoxins, types A and B (single and two-chain forms) to inactivate an intraneuronal component required for transmitter release were quantified in a phrenic-nerve-diaphragm preparation, cerebrocortical synaptosomes or the buccal ganglion of Aplysia californica and compared with the mouse toxicity assay. 2. Homogeneous preparations of the individually renatured polypeptide chains of both toxin types showed low residual toxicity in the whole animal and had no effect on neurotransmission in all three systems, when tested singly. 3. Mixtures of individually renatured heavy chain, from type A or B, and either light chain proved very effective in blocking the evoked release of acetylcholine when bath-applied to the buccal ganglion of Aplysia whilst they were relatively inactive on mammalian nerve terminals, indicating a less efficient uptake of the polypeptides in the latter. 4. When renatured together, the homologous, but not the heterologous, chains of each toxin type yielded toxic, disulphide-linked two-chain species. 5. A role for the heavy chain alone in acceptor recognition and membrane translocation was implicated by the blockade of acetylcholine release produced when light chain was applied to a ganglion of Aplysia previously bathed in heavy chain and washed extensively. No blockade was observed when the order of application of the two chains was reversed. 6. These findings are discussed in the context of the intracellular requirement for both the constituent toxin chains for toxicity, and in the apparent need for these chains to be linked via a disulphide bond for uptake in rodents but not in Aplysia.

Modification of the invariant tryptophan residue of two Naja naja neurotoxins

The invariant tryptophan residue of two cobra neurotoxins has been modified. The toxins were siamensis 3, the main neurotoxin of the venom of Naja naja siamensis (kaouthia) and siamensis 7C, a minor neurotoxic component of the same venom. Toxin 3 is of the structural type 71-5, 71 amino acid residues in a peptide chain crosslinked by five disulfides, and toxin 7C is of the type 62-4. Toxin 3 was treated with 2-hydroxy-5-nitrobenzyl bromide (Koshland's reagent) and three toxic derivatives were isolated by gel filtration on Sephadex G-50 and ion-exchange chromatography on Bio-Rex 70. Two of the derivatives had two substituent groups each and their LD 100 (intravenous)-doses were 500 and 300 μg/kg mouse. The third derivative with one substituent had an LD 100 of 200 μg/kg. The LD 100 of the native toxin is 100 μg/kg. The high residual toxicities (20–50%) indicate that the tryptophan residue is not a functionally essential group in a five-disulfide neurotoxin. This conclusion was confirmed by ozonization of toxin 3 and isolation as above of a derivative with an LD 100 of 200 μg/kg. Ozonization of toxin 7C gave a derivative with a residual activity of only 8%, or an LD 100 of 2000 μg/kg. The LD 100 of the native toxin is 150 μg/kg. In spite of this low residual toxicity, it can be concluded that tryptophan is not essential for the activity of a four-disulfide neurotoxin. The conclusion is based on three considerations: (1) The ozonized toxin 7C was completely separated from the native toxin, the toxicity of the ozonized form is thus not attributed to contamination with the native toxin. (2) The ozonized 7C is still a highly potent toxin, as toxic as (+)-tubocurarine chloride. The lethal doses expressed on a molar basis are 0.29 μmole/kg mouse for both substances. (3) The four- and five-disulfide neurotoxins are closely homologous proteins with the same target molecule, the nicotinic acetylcholine receptor. The active sites of the two types of toxins are probably similar. Since the active site of a five-disulfide neurotoxin does not include a tryptophan residue as a functional group, it is very unlikely that tryptophan should be functionally essential in a four-disulfide neurotoxin. The difference in residual toxicity between the ozonized toxins 3 and 7C is attributed to the difference in stability between the two types of toxins. A five-disulfide neurotoxin probably has a more rigid structure in the region of the invariant tryptophan owing to the presence of the extra disulfide bridge. A chemical modification might therefore cause greater structural changes with greater attendant loss of activity in a four-disulfide toxin as compared with a five-disulfide toxin.