Lethal Time Research Articles

Two novel entomopathogenic fungal species of Lecanicillium isolated from soil in China

The genus Lecanicillium was established in 2001 based on the type species Lecanicillium lecani (former, Verticillium lecani), which is an important entomopathogenic fungus. To date, more than thirty species in the genus have been reported, but much more are waiting to discover. In this study, two novel species isolated from soil in east China were identified. They differ from known closely related species primarily in sporulation structure, conidia and colony morphological characteristics, and sequence variations in the LSU, SSU, ITS, and TEF gene regions of the ribosomal DNA. Multigene phylogenetic analysis has provided strong molecular evidence supporting the classification of the strains, AH13B2 and JX15A210 within the genus Lecanicillium. The new species are formally named as follows: L. anqingense sp. nov. and L. renii sp. nov., respectively. Bioassays conducted on the greater wax moth, Galleria mellonella and crucifer aphid, Lipaphis erysimi showed that both fungal strains exhibit virulence. When treating with 1 × 108 spores/mL, the two strains gave mortality of > 60% and > 80% to G. mellonella and L. erysimi, respectively. Moreover, the median lethal times (LT50) of L. renii JX15A210 and L. anqingense AH13B2 against G. mellonella and L. erysimi were recorded as 8.06 d and 4.18 d, and 10.63 d and 5.07 d, respectively. This research enhances the species diversity within the genus Lecanicillium and provides valuable genetic resources for the biological control of pests and the development of biocontrol agents.

Possibilities of native endophytic fungi as entomopathogenic biocontrol agents at a local scale: the case of deciduous and non-deciduous Mediterranean forest trees

Tree endophytic fungi play an important role in reducing insect herbivory, either by repelling them or killing them directly. Identifying which fungi show such activity could lead to new environmentally friendly pesticides. In this study, the Mediterranean basin climate conditions are projected to harshen in the next decades, will increase vulnerability of tree species to pest invasions. Endophytic fungi were isolated from wood and leaves of Quercus pyrenaica, Q. ilex and Q. suber and tested for virulence against adults of the mealworm beetle, Tenebrio molitor L. using a direct contact method. Only 3 of 111 sporulating isolates had entomopathogenic activity, all identified as Lecanicillium lecanii. The pathogenicity of L. lecanii on T. molitor resulted in a median lethal time (TL50) of 14–16 d. Compared with commercial products, L. lecanii caused faster insect death than the nematode Steinernema carpocapsae and nuclear polyhedrosis virus (no effect on T. molitor survival), and slower than Beauveria bassiana (TL50 = 5), Beauveria pseudobassiana (TL50 = 8d) and Bacillus thuriengensis (80% mortality first day after inoculation). Mortality was also accelerated under water stress, reducing TL50 by an additional 33%. Remarkably, water stress alone had a comparable effect on mortality to that of L. lecanii isolates. This study confirms T. molitor as a good model insect for pathogenicity testing and agrees with management policies proposed in the EU Green Deal.Graphical abstract

Influence of temperature on growth, and sporulation of local EPF strains of <i>Penicillium</i> sp. and their efficacy against <i>Spodoptera litura</i> (Fabricius)

The research work was conducted to study the influence of temperature on the growth, and sporulation of local EPF strains of Penicillium spp. and their efficacy against Spodoptera litura (Fabricius) at Biological Control Laboratory, College of Agriculture, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli, Maharashtra - India. Observations were recorded on the effect of different temperature (20, 25, 30, 35, and 40°C) on varying patterns of radial growth and CFU count of Penicillium camponotum and Penicillium citrinum (Raper and Thom) and their efficacy against S. litura. The study found that both fungal species exhibited optimal radial growth and CFU count at 25 - 30°C. Specifically, P. camponotum displayed the highest cumulative radial growth (76.80 mm) and CFU count (452.80) at 30°C, whereas P. citrinum showed peak radial growth at 30°C (88.20 mm) and CFU count at 25°C (449.60) after 4 days, and 48 h of incubation, respectively. These conditions also correlated with the highest mortality rates of S. litura when treated with a concentration of 1 × 106 of Penicillium sp. strains, resulting in mortality percentages between 64.00 - 69.00 per cent at 7 days of post-treatment. Conversely, extreme temperatures such as 40°C drastically reduced fungal growth and efficacy, resulting in minimal mortality rates of 5.00 - 8.00 per cent. At 27 ± 3°C, both Penicillium strains showed the highest cumulative radial growth and CFU count, likely due to temperature fluctuation, resulting in increased cumulative mortality percentages against S. litura. The probit analysis of EPF strains P. camponotum, and P. citrinum revealed that LT50 and LT95 values varied significantly with temperature and culture type. For P. camponotum, the LT50 values from 5.4 - 24.3 days and, LT95 values ranged from 17.0 - 217.4 days, indicating substantial sensitivity to temperature (except 40°C). In contrast, P. citrinum showed narrower ranges of LT50 from 4.9 - 10.8 days, and LT95 values ranged from 14.5 - 46.6 days for all radial growth, and CFU count cultures at all tested temperatures except 40°C. These results demonstrate the significant influence of temperature on the radial growth and CFU count cultures, mortality percentage, and lethal time to cause 50 and 90 per cent mortality rates of S. litura.

Lethal and sublethal effects of flupyradifurone and cyantraniliprole on two neotropical stingless bee species.

Stingless bees are important pollinators in tropical regions, but their survival and behavior have been impacted by various factors, including exposure to insecticides. Here, we evaluated the lethal and sublethal effects of commercial formulations of two widely used insecticides, flupyradifurone (FPF formulation), and cyantraniliprole (CY formulation), on Melipona beecheii and Nannotrigona perilampoides. The study involved oral exposure of bees to insecticides, calculation of the lethal concentration (LC50) and the lethal time (LT50), and evaluation of walking and flight take-off activities. The LC50 values showed that the largest bee, M. beecheii, was more sensitive than N. perilampoides to both insecticides and that the FPF formulation had faster lethal effects in both species (N. perilampoides, 9.6 h; M. beecheii, 5 h) compared to the effects of the CY formulation (N. perilampoides, 17 h; M. beecheii, 24.7 h). Sublethal concentrations (LC50/10 and LC50/100) of both insecticides affected walking and flight take-off activities. After 6-24 h of exposure, both FPF and CY formulations significantly reduced the mean walking speed of N. perilampoides (0.962-1.402 cm/s) and M. beecheii (2.026-2.589 cm/s) compared to the control groups (N. perilampoides: 1.648-1.941 cm/s; M. beecheii: 2.759-3.471 cm/s). Additionally, the FPF and the CY formulation impaired individual flight take-off in both species. This study provides the first comprehensive evaluation of the lethal and sublethal effects of flupyradifurone and cyantraniliprole on M. beecheii and N. perilampoides, offering valuable information for future research on insecticide toxicity in stingless bees.

Fatty acids from soybeans: compatibility with Panonychus citri (Acari: Tetranychidae) and its two predators

Soybean oil is a significant alternative to synthetic chemicals for environmentally friendly and sustainable pest control. This study evaluated soybean oil and its fatty acids (palmitic, oleic, and stearic acids; individually) for their acaricidal and repellent effects against Panonychus citri (Tetranychidae) and its predators [Neoseiulus californicus and Neoseiulus barkeri [Phytoseiidae]) using laboratory foliar dipping and topical spray methods. The LC50 (Lethal Concentration) values for palmitic, oleic, stearic acids, and soybean oil against P. citri were 0.86%, 0.95%, 0.56%, and 0.05% for LDM (Leaf-dipping Method) and 11.92%, 5.34%, 1.46%, and 0.07% for TSM (Topical Spray Method). Soybean oil has maximum attraction (46.67 ± 3.6%) and significantly higher acaricidal effectiveness (46.25 ± 0.98%) compared to acids except oleic acid (50.83 ± 1.22%) against P. citri. Soybean oil showed the least toxicity, with the highest LT50 (Lethal Time) values for N. californicus (LDM: 1.19 h, TSM: 1.27 h). While soybean oil caused a less significant reduction in fertility and lower lethal efficacy (0.83 ± 0.83%) on predatory mites compared to P. citri. Compared to soybean oil, oleic acid reduces fertility rate (27 ± 1.60%) and is an effective repellent (65-75%) against N. californicus and N. barkeri and reduces prey consumption (15-16%) and laying eggs (0.75 ± 0.47) on treated surfaces compared to other treatments. Palmitic acid increases prey consumption (27.5 ± 3.28%) and fertility rates (5.00 ± 0.71) of N. californicus. Palmitic acid, despite being less toxic to P. citri, effectively repels predators, while soybean oil and oleic acid have stronger repellent effects. Leaf dipping was less effective than topical spray. Soybean oil, with its lower mortality rates and sublethal effects on reproduction and behavior, is a strong candidate for integrated pest management strategies. Oleic and palmitic acids, though effective, have repellency to beneficial predators.

Potential of Bacillus thuringiensis isolates to manage Gonipterus platensis (Coleoptera: Curculionidae) larvae populations.

The growing expansion of eucalyptus plantations in Brazil and the impact of exotic pests, such as Gonipterus platensis, demand effective, and sustainable biological control strategies. The aim of this study was to assess the pathogenicity of 10 Bacillus thuringiensis (Bt) isolates to neonate Gonipterus platensis larvae, commonly known as the eucalyptus weevil (Coleoptera: Curculionidae) with the specific focus of evaluating their potential to manage this pest while preserving its egg parasitoid, Anaphes nitens. To achieve this, the genomic DNA of the 10 Bt isolates was extracted using the thermal lysis method for molecular characterization of their Cry and Vip proteins. Neonate G. platensis larvae were subjected to bioassays with each isolate, at a concentration of 1 × 109 spores/ml, was applied on 10 larvae per replication (3 replications). The concentration and lethal time to kill 50% of the larvae were determined for the most effective isolates. The Bt isolates 107 and 178 isolates even at concentrations 10× lower than those recommended for commercial Bacillus thuringiensis var. tenebrionis (Btt) formulations against Coleoptera pests, achieved 100% mortality of G. platensis larvae. These isolates, with coleopteran-specific genes, caused high mortality of neonate Gonipterus platensis larvae. This indicates their potential for the biological control of this pest and maybe of other Coleoptera pests. Their use poses minimal risk to non-target organisms such as the egg parasitoid A. nitens and indicates a promising avenue for integrated pest management strategies with effective pest control while preserving the ecological balance.

Assessment of Lethal Dose and Lethal Time of Diazinon in Swiss Albino Mice

Approximately two thirds of deaths and millions of nonfatal cases are attributed to organophosphorus pesticides (OPP) annually. Diazinon, a prominent member of the OP pesticides, was introduced for both agriculture and public health usages during. For several decades, the compound was among the top pesticides in use in many countries, including Libya. The toxicity of this compound is based on its ability to inhibiting the acetylcholinesterase enzyme (AchE), which is vitally important for normal nerve impulse transmission. This study evaluated the acute toxicity of diazinon through acute exposure testing on female Swiss albino mice. In the acute toxicity test, 72 mice were subcutaneously injected with doses of diazinon ranging from 320 to 480 μL/ kg. Mortality and symptoms were recorded over four days, with the median lethal dose found to be 403.5 μL/kg. Based on the findings, the present study suggests that stringent controls and surveillance must be placed on diazinon and similar chemicals.

Metarhizium acridum transcription factor MaFTF1 negatively regulates virulence of the entomopathogenic fungus by controlling cuticle penetration of locusts.

The entomopathogenic fungus (EPF) Metarhizium acridum, a typical filamentous fungus, has been utilized for the biological control of migratory locusts (Locusta migratoria manilensis). Fungal-specific transcription factors (TFs) play a crucial role in governing various cellular processes in fungi, although TFs with only the Fungal_trans domain remain poorly understood. In this study, we identified a unique fungal-specific TF in M. acridum, named MaFTF1, which contains only a Fungal_trans domain and functions as a negative regulator of M. acridum virulence by influencing cuticle penetration. The virulence of the MaFTF1 knockout strain (ΔMaFTF1) against L. migratoria was increased, with a median lethal time (LT50) ~0.91 days shorter than that of the wild-type (WT) strain when inoculated topically, mimicking natural infection conditions. Correspondingly, ΔMaFTF1 penetrated the cuticle earlier than did the WT strain. Our investigation revealed that the development of appressoria was accelerated in ΔMaFTF1 compared with the WT strain. Furthermore, the appressoria of the ΔMaFTF1 displayed higher turgor pressure and an upregulated expression of fungal hydrolases active toward the insect cuticle. RNA sequencing analysis indicated that the differences in appressorium behavior between the strains were due to MaFTF1 regulating a complex metabolism pathway. This study revealed that MaFTF1 acts as a negative regulator of virulence, impacting the process of cuticle penetration by slowing the formation of appressoria, decreasing their turgor pressure, and reducing the expression of hydrolases in appressoria, revealing an unexpected strategy in the EPFs. © 2024 Society of Chemical Industry.

The potential of native strains of Metarhizium anisopliae (Metschnikoff) Sorokin in the management of brinjal ash weevil Myllocerus subfasciatus Guerin-Meneville (Curculionidae: Coleoptera)

ABSTRACT The ash weevil, Myllocerus subfasciatus, a major subterranean insect pest of brinjal, is managed using insecticides. Entomopathogenic fungi, such as Metarhizium anisopliae, have emerged as a viable alternative. To identify potential native Metarhizium spp., 38 soil samples were collected from brinjal fields. All eleven Metarhizium spp (anisopliae and robertsii) that were effective against M. subfasciatus had varying lethal concentrations and times. The M. anisopliae TNAU ENTMA TDM 8 strain was the most effective, with the lowest median lethal concentration of 2.13 × 105 conidia/ml and a median lethal time of 4.95 days. Field investigations still need to be conducted to identify potential candidate strains against ash weevil grubs and adults.

In Vitro Response of Two Strains of Cordyceps javanica to Six Chemical Pesticides.

To determine the compatibility of two new biocontrol fungi with common chemical pesticides, this study examined the effects of three insecticides, namely, avermectin, imidacloprid, and acetamiprid, and three fungicides, namely, chlorogenonil, boscalid, and kasugamycin, on the mycelial growth and spore germination of Cordyceps javanica strains IF-1106 and IJ-tg19. The insecticidal effects of mixed insecticides or fungicides with good compatibility with C. javanica IJ-tg19 against Myzus persicae were tested. The results showed that the six chemical pesticides exerted different degrees of inhibition on the mycelial growth of both C. javanica strains, with an obvious dose-dependent effect. The inhibitory effect of chlorothalonil on the mycelial growth of IF-1106 and IJ-tg19 was greater than 75%. Different kinds and concentrations of chemical pesticides had significant effects on spore germination. Among them, acetamiprid had little inhibitory effect on C. javanica spores. Therefore, the two C. javanica strains exhibited good compatibility with the insecticide acetamiprid and had some compatibility with avermectin and imidacloprid. Among the fungicides, the compatibility of the two strains of biocontrol fungi was the best with kasugamycin, followed by boscalid, while their compatibility with chlorothalonil showed the least compatibility. The median lethal time (LT50) of five concentrations of C. javanica IJ-tg19 (1 × 103, 1 × 104, 1 × 105, 1 × 106, and 1 × 107 spore/mL) mixed with acetamiprid against M. persicae were 5.28, 4.56, 3.80, 2.73, and 2.13 days, respectively, and the insecticidal rate was higher than that of fungus treatment alone (5.19, 4.59, 4.05, 3.32, and 2.94 days, respectively) or chemical pesticide treatment (5.36 days). This study provides data support and a theoretical basis for reducing the use of chemical pesticides, improving the efficiency of C. javanica-based insecticides, and optimizing the synergistic use of fungi and chemical pesticides.

Honey Bee Survival and Flight Capacity After Exposure to Sulfoxaflor Residues

Knowing the toxicity of insecticides used for pest control in melon on the bee Apis mellifera is essential for the conservation of this species in production areas, thus enabling the conciliation of insecticide applications with the sustainable use of pollinators in the field. From this perspective, this study aimed to evaluate the survival and flight ability of A. mellifera after exposure to residues of the insecticide Sulfoxaflor on melon leaves. The bioassay was conducted under laboratory conditions, in a completely randomized design set up in a 3 x 6 factorial arrangement, represented by two doses of the insecticide Sulfoxaflor (0.048 and 0.192 g i.a./L), one control (distilled water), and six exposure times after application of the product on melon leaves: immediately after spraying and 1h, 2h, 3h, 24h, and 48h after spraying. The insecticide Sulfoxaflor caused 100% morality in the bees exposed to the dose of 0.048 g i.a./L in the periods immediately after spraying and 1h, 2h, and 3h after spraying. The dose of 0.192 g i.a./L, regardless of the exposure time after spraying, resulted in 100% mortality. The median lethal time (TL50) provided by the Sulfoxaflor doses were significantly lower compared to the control, regardless of the time after foliar spraying. Sulfoxaflor negatively affected the flight ability of surviving bees. Regardless of the dose and exposure time after spraying, Sulfoxaflor was highly toxic via residues to A. mellifera under laboratory conditions.

Survival and Flight Capacity of Apis mellifera after Contact with Residues of Spiromesifen on Melon Leaves

Balancing the application of insecticides with the preservation of bees in agricultural areas poses a significant challenge to farmers. Therefore, this study aims to assess the survival and flight capacity of Apis mellifera (L.) bees after contact with residues of the insecticide/acaricide Spiromesifen on melon leaves. The experiment occurred under laboratory conditions, in a completely randomized design and a 4x3 factorial scheme, with two doses of Spiromesifen (0.24 and 0.288 g a.i./L), a positive control (Thiamethoxam), and an absolute control (distilled water), at three exposure times: 1, 2, and 3 hours after treatment application. Spiromesifen caused mortality of 15.33% and 13.80% after 1 hour of spraying, 20.05% and 17.72% after 2 hours of spraying, and 21.52% and 14.33% after 3 hours of spraying, for lower and higher doses, respectively. Spiromesifen provided a median lethal time of 112.9 hours, regardless of dose and exposure time. After exposure to the lower dose of Spiromesifen at 1, 2, and 3 hours after spraying, 28.41%, 8.0%, and 17.0% of the bees were able to fly, respectively, while at the higher dose, 33.55% and 8.11% were able to fly after 1 and 2 hours of spraying. Bees exposed after 3 hours of spraying at the higher dose showed a flight activity similar to the absolute control. Contact with Spiromesifen residues on melon leaves was slightly lethal to the honey bee A. mellifera, but it reduced the flight capacity of surviving bees after exposure.

CRISPR-Cas9-mediated enhancement of Beauveria bassiana virulence with overproduction of oosporein

Biocontrol agents play a pivotal role in managing pests and contribute to sustainable agriculture. Recent advancements in genetic engineering can facilitate the development of entomopathogenic fungi with desired traits to enhance biocontrol efficacy. In this study, a CRISPR-Cas9 ribonucleoprotein system was utilized to genetically improve the virulence of Beauveria bassiana, a broad-spectrum insect pathogen used in biocontrol of arthropod pests worldwide. CRISPR-Cas9-based disruption of the transcription factor-encoding gene Bbsmr1 led to derepression of the oosporein biosynthetic gene cluster resulting in overproduction of the red-pigmented dibenzoquinone oosporein involved in host immune evasion, thus increasing fungal virulence. Mutants defective for Bbsmr1 displayed a remarkable enhanced insecticidal activity by reducing lethal times and concentrations, while concomitantly presenting negligible or minor pleiotropic effects. In addition, these mutants displayed faster germination on the insect cuticle which correlated with higher density of free-floating blastospores in the hemolymph and accelerated mortality of the host. These findings emphasize the utility of genetic engineering in developing enhanced fungal biocontrol agents with customized phenotypic traits, and provide an efficient and versatile genetic transformation tool for application in other beneficial entomopathogenic fungi.

In vitro evaluation of the pathogenicity of fungi isolated from the Urabá region (Antioquia, Colombia) against Aedes aegypti larvae

Introduction. Aedes aegypti is an important vector of arboviral diseases like dengue among others. Traditional control strategies, such as the use of insecticides, have lost effectiveness due to the emergence of resistance in mosquito populations. Biological control and fungi applied for biocontrol are presented as viable and ecological alternatives. Objective. To evaluate in vitro pathogenicity of Trichoderma sp. isolates obtained from Urabá (Antioquia) on larvae of Ae. aegypti, and to determine the mean lethal concentration and mean lethal time of the most pathogenic isolate. Materials and methods. Using the sentinel larvae method of Ae. aegypti, fungi were isolated from water bodies in the Urabá region (Antioquia). The isolates were characterized morphologically and molecularly to determine their taxonomic identity. Pathogenicity tests were performed in vitro on Ae. aegypti larvae in the L2/L3 stages. Subsequently, a strain was selected to establish its mean lethal concentration and mean lethal time. Results. Trichoderma sp. strain AP-91 caused high mortality in larval populations of Ae. aegypti. We estimated a mean lethal concentration of 1.8 × 107 conidia/ml and a mean lethal time of 20.67 hours. Conclusion. The strain AP-91 showed potential for its use as biological control of Ae. aegypti, making it a suitable candidate for scale-up cultures applied to integrated vector management. This research suggests exploring compounds and enzymes produced by the AP-91 strain to understand better its pathogenicity.

Effect of microencapsulated/photoresistant extract of Azadirachta indica A. Juss seeds on Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctudae) and its persistence in semi-field

The development of bioinsecticides as an alternative to synthetic insecticides can benefit the economy of exporting and importing countries, family farming, production of high-quality commodities, food security, quality of life, and the natural environment. This study aimed to evaluate the effect of microencapsulated extracts of Azadirachta indica A. Juss on Spodoptera frugiperda J.E. Smith and their persistence in the plant under semi-field conditions. The fresh seeds of A. indica, collected in Brazil, were separated for the preparation of the microencapsulated dry extract (MDE) according to criteria registered in the patent. Lethal concentrations for S. frugiperda were determined using a dose response curve. The treatments were compared: MDE, control and positive control based on azadirachtin, evaluating mortality and growth inhibition. There was a significant difference in mortality between the treatments tested. The lethal concentrations LC50 and LC95, expressed in azadirachtin for the bioinsecticide, were 194.98 and 630.95 ppm, respectively. The growth inhibition of S. frugiperda was above 98 % in the treatments, except for the control. The MDE presented lethal times LT50 and LT90 lower than the other treatments (lethal time LT50 = 2.79 d; lethal time LT90 = 3.97 d). The MDE of A. indica seeds was effective in controlling S. frugiperda up to the fifth instar and is considered as slightly persistent to persistent depending on environmental conditions and irrigation, based on IOBC/WPRS parameters. The MDE has the potential to be a phytosanitary product approved for use in organic agriculture and with greater effectiveness in controlling S. frugiperda compared to similar pesticides.Abbreviations: BOD, Biochemical Oxygen Demand; CPA, commercial product based on azadirachtin; CLE, concentrated liquid extract; DAS, days after spraying; GI, growth inhibition; LC50, lethal concentration for 50% of the population; LC95, lethal concentration for 95% of the population; LT50, lethal time for 50% of the population; LT90, lethal time for 90% of the population; MDE, microencapsulated dry extract.

Evaluation of silver nanoformulated plant extracts against larvae of the fall armyworm, Spodoptera frugiperda (J. E. Smith), under laboratory and field conditions

AbstractThe fall armyworm, Spodoptera frugiperda (J. E. Smith), is a destructive pest of Zea mays (maize) and other agricultural crops. The synthetic insecticides predominantly used against this pest lead to pest resistance, environmental contamination and health hazards. This study evaluated nanoformulated aqueous extracts of some promising local plant species against third instar larvae of S. frugiperda under laboratory and field conditions. The initial screening bioassay showed highest larval mortality with a 20% extract of Nicotiana tabacum L. (66.67%), followed by Azadirachta indica A. Juss. (53.33%), Withania somnifera L. (46.67%), Melia azedarach L. (40%) and Dodonaea viscosa Jacq. (33.33%). The two most effective plant extracts (A. indica and N. tabacum) were further nanoformulated with silver nitrate (AgNO3) and bioassayed against S. frugiperda larvae using different concentrations. The results showed that these nanoformulated extracts caused significant larval mortality, with LC50 (lethal concentration that kills 50% of the population) and LT50 (lethal time to kill 50% of the population) values of 37.36 and 28.21% at 72 h, and 52.19 and 33.25 h at 80% concentration, respectively. Field experiments on Zea mays L. (maize) plants showed maximum larval reduction by nanoformulated A. indica extract (48%), followed by N. tabacum extract (36%), whereas 80% and 20% larval reduction was noted for the positive (SuperLock®, emamectin benzoate and tebufenozide) and the negative (water) controls, respectively. Furthermore, characterization of both silver nanoparticles‐based plant extract formulations was performed using ultraviolet–visible (UV–vis) spectroscopy, Raman spectroscopy and scanning electron microscopy (SEM) techniques, which confirmed the formation of silver nanoparticles. It is concluded that nanoformulated plant extracts can be an effective alternative to synthetic pesticides in combatting S. frugiperda and other lepidopteran pests.

Isolation and identification of native Chilean entomopathogenic fungi and their potential for the control of Drosophila suzukii

Drosophila suzukii is an invasive pest of berries and other soft-skinned fruits, was first detected in Chile in 2017, and has since spread over 2,800 km from north to south. Sustainable control of the spotted-wing drosophila (SWD) is essential due the negative attitude of the consumers toward the excessive use of insecticides. During a survey in Chile for biological control agents, thirty-two isolates of entomopathogenic fungi (EPF) were isolated from mycotized insects and soil samples, identified through sequence analysis, and tested against D. suzukii adults under laboratory conditions. The EPF identified are Akanthomyces muscarius, Beauveria bassiana, Beauveria pseudobassiana, Clonostachys rosea, Metarhizium alvesii, Metarhizium brunneum and Metarhizium robertsii. Six isolates caused 100 % of mortality of D. suzukii adults within ten days after the initial exposition to conidia; these included four B. bassiana isolates (LSB 110, LSB 114, LSB 122 and LSB 125), one M. robertsii isolate (LSB 115) and one M. brunneum isolate (LSB 127). LSB 122 and LSB 125 induced the shortest lethal time (LT50 4 and 4.2 days, respectively), while Akanthomyces and Clonostachys caused ≤ 36 % cumulative mortality of the adults at 10 days of exposure. These results indicate that isolates of B. bassiana, M. robertsii and M. brunneum have significant potential as microbial control agents against D. suzukii adults. This study marks a critical step forward in identifying and validating native entomopathogenic fungi in Chile for sustainable pest management. Future work will focus on further testing these isolates under laboratory, semi-field and field conditions to optimize their application in real-world agricultural settings.

Susceptibility of Aphis craccivora (Hemiptera: Aphididae) to three entomopathogenic Fusarium species

The black aphid (Aphis craccivora) is an insect pest that can cause significant losses to different agricultural crops. Entomopathogenic fungi can be good options for controlling this insect. Fusarium species have shown promising results in the biological control of several agricultural pests, mainly of the order Hemiptera. This study investigated the susceptibility of A. craccivora to 27 Fusarium isolates, distributed among F. sulawesiense (4), F. pernambucanum (6) and F. caatingaense (17). The viability of the conidia of all isolates was assessed by measuring their germination rate. Pathogenicity tests were conducted at 10⁷ conidia/mL, and the best-performing isolate was further tested at different concentrations (10⁴ to 10⁸ conidia/mL). Data were analyzed using ANOVA, Tukey's test at 5 %, and R for calculating lethal times (LT50,90) and lethal concentrations (LC50,90). All isolates had viable conidia with germination rates between 92.67 % and 100 %. Mortality rates ranged from 17.22 % to 90.23 %. F. pernambucanum URM 7559 had the shortest lethal times (LT50 of 2.24 days and LT90 of 4.42 days), followed by F. sulawesiense URM 7555 (LT50 of 2.35 days and LT90 of 4.77 days) and F. caatingaense with LT50 of 3.93 days for URM 6784 and LT90 of 8.27 days for URM 6807. The three Fusarium species tested, especially F. pernambucanum, showed promise in the biological control of A. craccivora. Although the results are promising, additional studies are needed to evaluate the safety, field efficacy and environmental impacts of Fusarium use, focusing on the interaction with the agricultural ecosystem and the risks to non-target organisms.

Conserved fungal effector NLS1 suppresses Lepidoptera insect immunity by targeting the host defense protein Hdd11.

Entomopathogenic fungi have been widely used as the main mycoinsecticide for controlling agricultural and forest pests. The effector molecules of these mycopathogens have evolved to adapt to their hosts. The role of fungal effectors in evading the host immune system in insects remains mainly unclear. We characterized the widely distributed fungal effector necrosis-inducing-like secreted protein 1 (NLS1) in the entomopathogenic fungus Metarhizium robertsii. Our findings revealed the presence of M. robertsii NLS1 (MrNLS1) in host hemocytes during the early stage of hemocoel infection. MrNLS1 knock down (ΔMrNLS1) reduced fungal pathogenicity during infection and altered the expression of host immune genes. The molecular docking results and the yeast 2-hybrid assay confirmed that MrNLS1 interacts with the host defense protein Hdd11. The phylogenetic analysis indicated that Hdd11 is conserved across a broad range of Lepidoptera species. Knock down of hdd11 in Helicoverpa armigera, Bombyx mori, and Galleria mellonella markedly suppressed their immune responses against M. robertsii. However, no significant difference was observed in the mean lethal time between hdd11-knockdown Lepidoptera species infected with ΔMrNLS1 and those infected with wild-type M. robertsii. Therefore, in Lepidoptera insects, Hdd11 is essential for fungal defense. In conclusion, M. robertsii infects Lepidoptera insects by targeting host Hdd11 through its protein MrNLS1, thereby suppressing the host immune response. Our findings clarify the molecular mechanisms underlying fungal infection pathogenesis.

Differences in seasonal dynamics and pyrethroid resistance development among Anopheles Hyrcanus group species

BackgroundThe Anopheles Hyrcanus group, which transmits Plasmodium vivax, consists of six confirmed species in South Korea. An epidemiological study revealed differences in the seasonal occurrence patterns of each species. Pyrethroid resistance in An. sinensis dates back to the early 2000s, whereas information on pyrethroid resistance in other species is lacking despite their greater significance in malaria epidemiology.MethodsAnopheles mosquitoes were collected from two malaria-endemic regions in South Korea for 2 years and their knockdown resistance (kdr) mutations were genotyped. The larval susceptibility to λ-cyhalothrin was compared in six Anopheles species and its seasonal changes in three species were investigated. The full-length sequences of the voltage-sensitive sodium channel (VSSC) were compared across six species to evaluate potential target-site insensitivity. The contribution of the kdr mutation to phenotypic resistance was confirmed by comparing median lethal time (LT50) to λ-cyhalothrin between populations of Anopheles belenrae with distinct genotypes.ResultsThe composition and seasonal occurrence of rare species (Anopheles kleini, Anopheles lestri, and Anopheles sineroides) varied considerably, whereas An. sinensis occurs continuously throughout the season. A kdr mutation in the form of heterozygous allele was newly identified in An. belenrae, An. lesteri, An. pullus, and An. sineroides. The baseline susceptibility to λ-cyhalothrin was the highest in An. belenrae, followed by An. lesteri, An. sineroides, An. kleini, An. pullus, and An. sinensis, with median lethal concentration (LC50) values ranging from 6.0- to 73.5-fold higher than that of An. belenrae. The susceptibility of An. sinensis and An. pullus varied by season, whereas that of An. belenrae remained stable. The kdr-heterozygous An. belenare population exhibited 5.1 times higher LT50 than that of the susceptible population. Species-specific VSSC sequence differences were observed among the six species.ConclusionsOur findings suggest that the status and extent of pyrethroid resistance vary among Anopheles Hyrcanus group species. While An. sinensis, the predominant species, developed a considerable level of pyrethroid resistance through kdr mutation, the resistance levels of other species appeared to be less pronounced. Large-scale monitoring is crucial to fully understand species-specific seasonal occurrence and resistance status for effective management strategies, considering the ongoing impact of climate change on their vectorial capacity.Graphical