Direct Contact Bioassays Research Articles

Nematicidal Activity of Volatiles against the Rice Root-Knot Nematode and Environmental Safety in Comparison to Traditional Nematicides.

The rice root-knot nematode (RRKN), Meloidogyne graminicola Golden and Birchfield 1965, is a dangerous crop pest that affects rice production on a global scale. The largest rice-producing countries struggle with the impacts of RRKN infestation, namely, underdeveloped plants and a reduction in rice grain that can reach up to 70% of crop yield. In addition, the shift to strategies of sustainable pest management is leading to a withdrawal of some of the most effective pesticides, given the dangers they pose to the environment and human health. Volatile metabolites produced by plants can offer safer alternatives. The present study characterized the nematicidal activity of volatile phytochemicals against the RRKN and compared the most active with commercial nematicides concerning their safety to the environment and human health. Rice plants were used to grow large numbers of RRKNs for direct-contact bioassays. Mortality induced by the volatiles was followed for four days on RRKN second-stage juveniles. Of the 18 volatiles tested, carvacrol, eugenol, geraniol, and methyl salicylate showed the highest mortalities (100%) and were compared to traditional nematicides using (eco)toxicological parameters reported on freely available databases. While methyl salicylate had a faster activity, carvacrol had more lasting effects. When compared to synthetic nematicides, these volatile phytochemicals were reported to have higher thresholds of toxicity and beneficial ecotoxicological parameters. Ultimately, finding safer alternatives to traditional pesticides can lower the use of damaging chemicals in farming and leverage the transition to agricultural practices with a lower impact on biodiversity.

Essential Oil Volatiles as Sustainable Antagonists for the Root-Knot Nematode Meloidogyne ethiopica

Climate change is prompting a shift of tropical pests to locations with a previously lower probability of invasion. This is the case for root-knot nematodes, Meloidogyne sp., particularly of the tropical group. Among them, M. ethiopica is now considered a threat to European food security. The development of novel sustainable nematicides can be based on in vitro bioassays of highly active phytochemicals, e.g., volatiles from essential oils. However, a steady supply of nematodes is often very difficult and dependent on environmental conditions. In the present study, an in vitro co-culture system of M. ethiopica parasitizing hairy roots of Solanum lycopersicum was established, for the first time, to easily obtain populations of second-stage juveniles (J2). These were then used to screen the nematicidal activity of 10 volatile compounds characteristic of essential oils. Finally, information on the most successful compounds was reviewed to predict their environmental dispersion and ecotoxicological hazards. The M. ethiopica population obtained from the co-culture was morphologically similar to reported populations in natural conditions and could be accurately used in direct-contact bioassays. The aldehydes citral and citronellal induced complete mortality of the tested J2, at 1 mg/mL, while compounds from other chemical groups were not as successful. In comparison to commonly used commercial nematicides, citral and citronellal were less likely to accumulate in the water environmental compartment and have lower reported toxicities compared to aquatic organisms and to mammals. Overall, in vitro co-cultures showed the potential to expedite the screening and discovery of bioactive compounds as a contribution to the development of sustainable biopesticides, as well as to lower the impacts of modern farming on agroecosystems.

Assessing the toxicity of contaminated soils via direct contact using a gas production bioassay of thiosulfate utilizing denitrifying bacteria (TUDB)

A direct contact bioassay of thiosulfate utilizing denitrifying bacteria (TUDB) based on inhibition of gas production was deployed to assess the toxicity of naturally contaminated field soils and soils artificially contaminated with heavy metals. Test procedure producing optimal conditions responsible for maximum gas production was 0.5 mL test culture, 1 g soil sample, 80 RPM, and 48 h reaction time. Similarly, the concentrations which generated a 50% reduction in gas production by TUDB for the tested heavy metals were 3.01 mg/kg Cr6+; 15.30 mg/kg Ni2+;15.50 mg/kg Cu2+;16.60 mg/kg Ag+; 20.60 mg/kg As3+; 32.80 mg/kg Hg2+; 54.70 mg/kg Cd2+; and 74.0 mg/kg Pb2+. Because soil toxicity is usually influenced by various physicochemical characteristics, ten reference soils were used to determine the toxicity threshold for evaluating the toxicity of naturally contaminated field soils. All eight contaminated soils were toxic to the TUDB bioassay because their levels of inhibition ranged between 72% and 100% and exceeded the determined toxicity threshold of 10%. Compared to other direct contact assays, the newly developed assay TUDB proved to be very robust, producing highly sensitive data while the different soil physicochemical properties exerted minimal influence on the gas production activity of TUDB. Additionally, the simplicity of the developed methodology coupled with the elimination of pretreatment procedures such as elutriation, and ability to perform generate sensitive data in turbid and highly colored samples makes it, cost-effective, and easily adaptable for the assessment of heavy metal and field contaminated soils when compared with other conventional assays which require sophisticated instrumentation and prolonged testing procedures and times.

A direct contact bioassay using immobilized microalgal balls to evaluate the toxicity of contaminated field soils

The present study used a bioassay of immobilized microalgae (Chlorella vulgaris) via direct contact to assess the toxicity of eleven uncontaminated (reference) and five field contaminated soils with various physicochemical properties and contamination. Photosynthetic oxygen concentration in the headspace of the test kit by Chlorella vulgaris in the reference soils ranged between 12.93% and 14.80% and only 2.54%–7.14% in the contaminated soils, respectively. Inherent test variability (CVi) values ranged between 2.90% and 9.04%; variation due to soil natural properties (CVrs) ranged between 0.33% and 13.0%; and minimal detectable difference (MDD) values ranged from 4.69% to 11.6%. A computed toxicity threshold of 15% was established for microalgae soil toxicity tests based on calculations of the maximal tolerable inhibition (MTI). All contaminated soils were considered toxic to microalgae because their levels of inhibition ranged between 39.5% and 82.9%, exceeding the 15% toxicity threshold. It can be concluded that the elevated concentrations of heavy metals and organic contaminants in the contaminated soils induced the higher inhibitory levels. Overall, direct contact soil toxicity tests using immobilized microalgae provided coherent and repeatable data and can be utilized as a simple and suitable tool for the toxicity testing of contaminated field soils.

A Direct Contact Bioassay Using Immobilized Microalgal Balls to Evaluate the Toxicity of Contaminated Field Soils

A Direct Contact Bioassay Using Immobilized Microalgal Balls to Evaluate the Toxicity of Contaminated Field Soils

Nematicidal Activity of Essential Oils on a Psychrophilic Panagrolaimus sp. (Nematoda: Panagrolaimidae).

Essential oils (EOs) have historically been used for centuries in folk medicine, and nowadays they seem to be a promising control strategy against wide spectra of pathogens, diseases, and parasites. Studies on free-living nematodes are scarce. The free-living microbivorous nematode Panagrolaimus sp. was chosen as the test organism. The nematode possesses extraordinary biological properties, such as resistance to extremely low temperatures and long-term survival under minimal metabolic activity. Fifty EOs from 22 plant families of gymnosperms and angiosperms were tested on Panagrolaimus sp. The aims of this study were to investigate the in vitro impact of EOs on the psychrophilic nematode Panagrolaimus sp. in a direct contact bioassay, to list the activity of EOs based on median lethal concentration (LC50), to determine the composition of the EOs with the best nematicidal activity, and to compare the activity of EOs on Panagrolaimus sp. versus plant parasitic nematodes. The results based on the LC50 values, calculated using Probit analysis, categorized the EOs into three categories: low, moderate and highly active. The members of the laurel family, i.e., Cinnamomum cassia and C. burmannii, exhibited the best nematicidal activity. Aldehydes were generally the major chemical components of the most active EOs and were the chemicals potentially responsible for the nematicidal activity.

A solid-phase direct contact bioassay using sulfur-oxidizing bacteria (SOB) to evaluate toxicity of soil contaminated with heavy metals

The current study tested the employment of sulfur-oxidizing bacteria (SOB) in a direct contact bioassay to evaluate toxicity of soil contaminated with heavy metals. SOB were directly exposed to soil spiked with arsenic, nickel, hexavalent chromium, zinc, mercury, copper, lead, or cadmium for 12 h in a kit-type bioassay. Oxygen consumption by SOB was used as the end-point for toxicity assessment. The results demonstrate that oxygen consumption by SOB decreased as the doses of spiked heavy metals increased. Oxygen consumption in the tests spiked with arsenic, nickel, hexavalent chromium, or zinc was less than that spiked with mercury, copper, lead, or cadmium at the same concentrations. The 12 h half-maximum effective concentrations (EC50) of SOB for arsenic, nickel, hexavalent chromium, zinc, mercury, copper, lead, and cadmium were analyzed to be 12.7, 13.3, 19.1, 20.8, 40.1, 67.2, 101.5, and 105.4 mg/kg, respectively. These EC50 values are largely within the regulatory standards of heavy metals in agricultural soil, suggesting that the SOB kit test is sufficiently sensitive for use in on-site soil toxicity tests. The findings from the current study show that the SOB kit test is a suitable tool for evaluating heavy metal-induced toxicity of soil.

Efficacy of Insecticides Against Spodoptera frugiperda (Smith, 1797)

The Brazil’s economy is supported by agribusiness, however, the continuous cycle of food production and favorable climate contributes to the incidence of pest insects all year round. The fall armyworm Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) is considered the main insect in the corn crop, due to voracity of the caterpillars and occurrence throughout the crop cycle. Therefore, the chemical control has been demanded considerably, causing emergence of populations resistant to the different products, as well as implications in the environment. Thus, studies are needed to evaluate the efficiency of chemical insecticide control according to the susceptibility and the stages of biological development of the pest. The objective of this work was to evaluate the performance of isolated and combined insecticides for the control of S. frugiperda caterpillars under laboratory conditions. In the residual and direct contact bioassays, 8 treatments with 5 replications were used in a completely randomized design, performed with 2nd and 5th instar. The results showed that by residual contact after 72 hours, clofenapir + zeta-cypermethrin treatment had 100% efficacy in the mortality of both instars. When applied via direct contact 72 hours later, the combined treatments showed an efficiency above 80%. However, for an integrated pest management program, where it was recommended the association of different control tactics, the management of insecticides with clofenapir + zeta-cypermethrin was effective with 100% control efficiency in both instars.

Insecticides Management Used in Soybean for the Control of Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Eribidae)

The soybean is the main agricultural product produced in Brazil. Among the States, Rio Grande do Sul was the third largest producer in the 2017/2018 harvest. However, insects interfere on the productivity and provide an increase in the cost of production. The velvetbean caterpillar, Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Eribidae) is considered one of the main leaf stripper caterpillars. The occurrence can completely fade, reflected in the grain yield. The chemical method is the main control adopted, however, due to the inadequate management of the crop, failures are observed frequently on the field. In this context, the insecticide management is considered an alternative, still little adopted, due to a lack of knowledge between the movement action and the susceptibility of the insect. Therefore, the objective was to evaluate the performance of insecticides used in the soybean crop for the control of A. gemmatalis under laboratory conditions. In the residual and direct contact bioassays, eight treatments with five replicates were used in a completely randomized design, with 2nd and 5th instar caterpillars of A. gemmatalis. The results showed that by residual contact during 24 hours, all the treatments with exception of the control had effectiveness in both instar mortality. The treatments flubendiamide + zeta-cypermethrin and zeta-cypermethrin after four hours application reached 100% of effectiveness. Thus, aiming an integrated pest management program, where it advocated the association of different control tactics, the insecticide management with flubendiamide + zeta-cypermethrin were effective with 100% of control efficiency in both instars.

Biomarkers in Tubifex tubifex for the metalaxyl and metalaxyl-M toxicity assessment in artificial sediment.

Efficacy and potential environmental risks should be considered when applying enantiopure pesticides. In this study, Tubifex tubifex and its oxidative stress biomarkers were assessed for the toxicity of metalaxyl and metalaxyl-M in sediment. The toxicity assessment was conducted with artificial sediment and reconstituted water. Five test concentrations (from 0 to 100mg/kg) were set for the short-term (4days) exposure. The long-term (28days) exposure was conducted with the environmental concentration (from 0.5 to 5mg/kg). For the short-term exposure, the increase of superoxide dismutase (SOD) activity was observed, and a clear concentration-response relationship was found in the metalaxyl treatments. The decrease of catalase (CAT) and glutathione peroxidase (GPx) activity could be caused by oxidative stress. The decrease of glutathione (GSH) content and the increase of glutathione-S-transferase (GST) might be due to antioxidation defense and detoxification mechanisms. The increase of malondialdehyde (MDA) might be due to the saturation of antioxidant systems and the accumulation and toxicity of contaminations. In the long-term exposure, the changes of biomarkers in T. tubifex reflected the oxidative stress and detoxification metabolisms. GSH and the related enzymes were important in detoxification processes and involved in the oxidative stress in toxicity mechanism. The long-term direct contact bioassay is sensitive and appropriate to reflect the lower concentration of contaminants.

Direct nematicidal effects of methyl jasmonate and acibenzolar-S-methyl against Meloidogyne incognita

The aim of this study was to examine the nematicidal properties of two defence inducers against the root-knot nematode Meloidogyne incognita. A direct-contact bioassay was applied to evaluate the nematicidal effects of acibenzolar-S-methyl (ASM) and methyl jasmonate (MEJA) on second-stage juveniles (J2). Nematodes were incubated in different concentrations of these compounds, and the numbers of immobile nematodes were counted after 24 and 48 h post incubation. Tap water was then added to verify whether the nematodes recovered or remained dead at 72 h. The percentage of dead nematodes was used as indicator for the toxicity of the different solutions. Our results show that ASM, in the formulation of Bion®, and MEJA have nematicidal properties.

First report on Meloidogyne chitwoodi hatching inhibition activity of essential oils and essential oils fractions

The Columbia root-knot nematode (CRKN), Meloidogyne chitwoodi, is an EPPO A2 type quarantine pest since 1998. This nematode causes severe damage in economically important crops such as potato and tomato, making agricultural products unacceptable for the fresh market and food processing. Commonly used nematicidal synthetic chemicals are often environmentally unsafe. Essential oils (EOs) may constitute safer alternatives against RKN. EOs, isolated from 56 plant samples, were tested against CRKN hatching, in direct contact bioassays. Some of the most successful EOs were fractionated and the hydrocarbon molecules (HM) and oxygen-containing molecules (OCM) fractions tested separately. 24 EOs displayed very strong hatching inhibitions (≥90 %) at 2 µL mL−1 and were further tested at lower concentrations. Dysphaniaambrosioides, Filipendula ulmaria, Ruta graveolens, Satureja montana and Thymbra capitata EOs revealed the lowest EC50 values (<0.15 µL mL−1). The main compounds of these EOs, namely 2-undecanone, ascaridol, carvacrol, isoascaridol, methyl salicylate, p-cymene and/or γ-terpinene, were putatively considered responsible for CRKN hatching inhibition. S. montana and T. capitata OCM fractions showed hatching inhibitions higher than HM fractions. The comparison of EO and corresponding fractions EC50 values suggests interactions between OCM and HM fractions against CRKN hatching. These species EOs showed to be potential environmentally friendly CRKN hatching inhibitors; nonetheless, bioactivity should be considered globally, since its HM and OCM fractions may contribute, diversely, to the full anti-hatching activity.

Laboratory and Simulated Field Bioassays to Evaluate Larvicidal Activity of Pinus densiflora Hydrodistillate, Its Constituents and Structurally Related Compounds against Aedes albopictus, Aedes aegypti and Culex pipiens pallens in Relation to Their Inhibitory Effects on Acetylcholinesterase Activity.

The toxicity of Pinus densiflora (red pine) hydrodistillate, its 19 constituents and 28 structurally related compounds against early third-instar larvae of Aedes albopictus (Ae. albopictus), Aedes aegypti (Ae. aegypti) and Culex pipiens palles (Cx. p. pallens) was examined using direct-contact bioassays. The efficacy of active compounds was further evaluated in semi-field bioassays using field-collected larval Cx. p. pallens. Results were compared with those of two synthetic larvicides, temephos and fenthion. In laboratory bioassays, Pinus densiflora hydrodistillate was found to have 24 h LC50 values of 20.33, 21.01 and 22.36 mg/L against larval Ae. albopictus, Ae. aegypti and Cx. p. pallens respectively. Among the identified compounds, thymol, δ-3-carene and (+)-limonene exhibited the highest toxicity against all three mosquito species. These active compounds were found to be nearly equally effective in field trials as well. In vitro bioassays were conducted to examine the acetylcholinesterase (AChE) inhibitory activity of 10 selected compounds. Results showed that there is a noticeable correlation between larvicidal activity and AChE inhibitory activity. In light of global efforts to find alternatives for currently used insecticides against disease vector mosquitoes, Pinus densiflora hydrodistillate and its constituents merit further research as potential mosquito larvicides.

&lt;I&gt;Ricinus communis&lt;/I&gt; and &lt;I&gt;Jatropha curcas&lt;/I&gt; (Euphorbiaceae) Seed Oil Toxicity Against &lt;I&gt;Atta sexdens rubropilosa&lt;/I&gt; (Hymenoptera: Formicidae)

Leaf-cutting ants are the main herbivores in the New World tropics. Although the toxicity of seed oils against these ants has been poorly investigated, previous results revealed that seed oils exert considerable toxic activity against these insects. This paper analyzes the toxic action and deterrent properties of castor oil, Ricinus communis L., and physic nut oil, Jatropha curcas L., against workers of the leaf-cutting ant Atta sexdens rubropilosa reared in laboratory. Toxic effect was analyzed by feeding insects artificial diets supplemented with different oil concentrations and direct contact with the two oils. Deterrent activity was assessed by measuring the frequency of attendance to diets during the first 48 h of the ingestion bioassay. Castor oil at 10 and 30 mg/ml and physic nut oil at 5, 10, and 30 mg/ml were toxic by ingestion. In the direct contact bioassay, toxicity was observed for physic nut oil at 0.1 and 0.2 mg/ml, whereas castor oil exerted toxic effects only when the highest concentration was applied. Also, castor oil had a more pronounced deterrent effect against the leaf-cutting ant, compared with physic nut oil. Methods to apply these oils to control these insects are discussed.

Susceptibility of Megachile rotundata to insecticides used in wild blueberry production in Atlantic Canada

The alfalfa leafcutting bee, Megachile rotundata (Fabricius), is a valuable wild and managed pollinator of lowbush blueberry (syn. ‘wild blueberry’, Vaccinium angustifolium Ait.), in Atlantic Canada. As some insecticides may present a hazard to pollinators, we assessed the susceptibility of M. rotundata to insecticides used or projected for future use in lowbush blueberry pest management. In topical direct contact bioassays, adults were susceptible to phosmet, spinosad, spinetoram, and deltamethrin. Based on findings from these laboratory studies, it appears that when used at recommended or projected application rates, each of these compounds poses a hazard to M. rotundata by direct contact. In a second experiment, eggs and larvae were collected in the field and their pollen provisions were treated with deltamethrin, flubendiamide, and spinetoram at field relevant concentrations. Larvae treated with deltamethrin and spinetoram in the laboratory either died before spinning a cocoon or, in the case of spinetoram, occasionally pupated without spinning a cocoon. Flubendiamide was not toxic to adult M. rotundata by direct contact and had no effect on larval survivorship, or time to complete cocoon spinning. Emergence after overwintering was relatively poor overall, but there was no effect of treatment. Based on these results, flubendiamide appears safe to use in the presence of M. rotundata, whereas the other insecticides we tested may pose a hazard.

Insecticidal and repellent activities of insecticide-sucrose solutions to Culex pipiens molestus (Diptera: Culicidae) under laboratory and field conditions

Culex pipiens molestus Forskal has been reported as a dominant species in underground structures of urban areas in the Republic of Korea (ROK) during all seasons and becomes bothersome to humans in late autumn and winter. Most Cx. pipiens molestus in septic tanks are controlled in the ROK using larvicides such as Bt and IGR. However, there are a number of problems associated with larvicides, such as high cost and requirement for frequent use. In the present work, a new control method for Cx. pipiens molestus in septic tanks by using mixtures of sucrose solution with insecticides was investigated. The insecticidal and repellent activities of ten insecticides were evaluated for best control of Cx. pipiens molestus in septic tanks. Firstly, differences in susceptibilities to insecticides were evaluated in topical assays by forced direct contact bioassay and in a screened wire cage by free direct contact bioassay. The difference in insecticide susceptibility in the mosquitoes was the result of repellency by the insecticides. In three septic tanks, the density of Culex mosquitoes was sharply reduced by a deltamethrin-sucrose solution kit. The results demonstrated the potential for mosquito control by deltamethrin-sucrose solution, and the study offers basic information related to mosquito control in septic tanks.

Estabilidade da resistência de Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae) a hexythiazox em pomares de citros

The false spider mite, Brevipalpus phoenicis (Geijskes), is one the most important pests of the citrus groves and transmits the citrus leprosis virus. The acaricide hexythiazox has been widely used for controlling B. phoenicis in citrus groves. The resistance of this species to hexythiazox has already been detected at high frequencies at some locations. In order to implement a resistance management program, studies were undertaken to understand the stability of the resistance of B. phoenicis to hexythiazox by 1) comparing the life-history of susceptible (S) and resistant (R) strains under laboratory conditions, and 2) evaluating the dynamics of hexythiazox resistance in citrus field plots with low (< 20%) and high (> 60%) frequency of resistance, during two years. The frequencies of resistance were estimated with direct contact bioassays on eggs with discriminating concentration of 18 mg of hexythiazox/L of water. There were no significant differences between S and R strains, based on biological parameters evaluated to build fertility life tables. However, the resistance of B. phoenicis to hexythiazox was unstable under field conditions; that is, significant reductions in the frequency of resistance were observed in the absence of selection pressure, either in citrus field plots with low or high frequency of resistance. Therefore, the instability of B. phoenicis resistance to hexythiazox can be exploited in resistance management programs.

Resistance to hexythiazox in Brevipalpus phoenicis (Acari: Tenuipalpidae) from Brazilian citrus.

The objective of this study was to collect baseline information for implementing an acaricide resistance management program of Brevipalpus phoenicis (Geijskes) to hexythiazox in Brazilian citrus groves. The egg susceptibility of B. phoenicis to hexythiazox was measured by a direct contact bioassay. The estimated LC50 for the S strain was 0.89 mg hexythiazox L(-1) of water (95% FL 0.75-1.03). After hexythiazox selection of a field-collected population associated with intense hexythiazox use, a resistance ratio greater than 10,000-fold was detected. Results from a survey revealed a great variability in the frequency of resistance in populations of B. phoenicis collected from citrus groves located in the State of São Paulo. No relationship was observed between the intensity of hexythiazox use and the frequency of resistance. Studies on dynamics of resistance showed that the resistance of B. phoenicis to hexythiazox is stable under laboratory conditions. Therefore, there is an urgent need to implement resistance management of B. phoenicis to hexythiazox in order to prolong its effective use in Brazilian citrus groves.