Effects of fungicide treatments on mycorrhizal communities and carbon acquisition in the mixotrophic Pyrola japonica (Ericaceae).

Pyrola japonica, a member of the family Ericaceae, is a mixotroph that grows on forest floors and obtains carbon (C) from both its photosynthesis and its mycorrhizal fungi. Its mycorrhizal community is dominated by Russulaceae. However, the mechanism of its C acquisition and its flexibility are not well understood. Our aim was to assess the impact of disturbance of the mycorrhizal fungal communities on C acquisition by P. japonica. We repeatedly applied a fungicide (Benomyl) to soils around P. japonica plants in a broad-leaved forest of central Japan, in order to disturb fungal associates near roots. After fungicide treatment, P. japonica roots were collected and subjected to barcoding by next-generation sequencing, focusing on the ITS2 region. The rate of mycorrhizal formation and α-diversity did not significantly change upon fungicide treatments. Irrespective of the treatments, Russulaceae represented more than 80% of the taxa. Leaves and seeds of the plants were analysed for 13C stable isotope ratios that reflect fungal C gain. Leaf and seed δ13C values with the fungicide treatment were significantly lower than those with the other treatments. Thus the fungicide did not affect mycorrhizal communities in the roots, but disturbed mycorrhizal fungal pathways via extraradical hyphae, and resulted in a more photosynthetic behaviour of P. japonica for leaves and seeds.

Electrochemical determination of benomyl using MWCNTs interspersed graphdiyne as enhanced electrocatalyst.

Graphdiyne (GDY) has attracted a lot of interest in electrochemical sensing application with the advantages of a large conjugation system, porous structure, and high structure defects. Herein, to further improve the sensing effect of GDY, conductive MWCNTs were chosen as the signal accelerator. To get a stable composite material, polydopamine (PDA) was employed as connecting bridge between GDY and MWCNTs-NH2, where DA was firstly polymerized onto GDY, followed by covalently linking MWCNTs-NH2 with PDA through Michael-type reaction. The formed GDY@PDA/MWCNTs-NH2 composite was then explored as an electrochemical sensor for benomyl (Ben) determination. GDY assists the adsorption and accumulation of Ben molecules to the sensing surface, while MWCNTs-NH2 can enhance the electrical conductivity and electrocatalytic activity, all of which contributing to the significantly improved performance. The proposed sensor displays an obvious oxidation peak at 0.72 V (vs. Hg|Hg2Cl2) and reveals a wide linear range from 0.007 to 10.0 µM and a low limit of detection (LOD) of 1.8 nM (S/N = 3) toward Ben detection. In addition, the sensor shows high stability, repeatability, reproducibility, and selectivity. The feasibility of this sensor was demonstrated by detecting Ben in apple and cucumber samples with a recovery of 94-106% and relative standard deviations (RSDs) less than 2.3% (n = 5). A sensitive electrochemical sensing platform was reported for benomyl (Ben) determination based on a highly stable GDY@PDA/MWCNTs-NH2 composite.

Monitoring Benzimidazole Resistance in Phyllosticta citricarpa Using a Molecular Assay Targeting Mutations in Codons 198 and 200 of the ß-Tubulin Gene.

Citrus black spot (CBS), caused by Phyllosticta citricarpa, is an economically important disease, which is effectively controlled by repeated fungicide applications to protect fruit from infection. Systemic fungicides such as benzimidazoles are widely used for controlling CBS in South Africa, but the molecular mechanisms of benzimidazole resistance in P. citricarpa had not been investigated. Analysis of the nucleotide sequence of the ß-tubulin gene in P. citricarpa revealed mutations inducing three amino acid replacements in benzimidazole-resistant isolates when compared with those of sensitive strains. Amino acid replacements in benzimidazole-resistant isolates included the change of glutamic acid to either alanine or lysine at codon 198 of the ß-tubulin gene and the change from phenylalanine to tyrosine at codon 200. All three mutations were previously implicated in benzimidazole resistance in several fungal pathogens. A PCR assay was designed to amplify a portion of the ß-tubulin gene, which is subsequently sequenced to identify benzimidazole resistance in P. citricarpa. This PCR and sequence assay was found to be a more rapid and reliable method for detecting resistance compared with the fungicide-amended plate tests and is valuable for monitoring the occurrence of benzimidazole-resistant P. citricarpa and for assessment of the need for alternative CBS management practices.

2D Leaf-Like Structured ZIF-L Embedded Electrochemically Reduced Graphene Oxide Composite as an Electrochemical Sensing Platform for Sensitively Detecting Benomyl.

In this work, a two-dimensional leaf-like framework-L embedded electrochemically reduced graphene oxide (ERGO@ZIF-L) was proposed as an outstanding electrode material for the sensitive electrochemical sensing of benomyl (BM). ZIF-L is surrounded by ERGO, which could effectively ensure the stability and dispersion of ZIF-L. With this unique combination, the prepared ERGO@ZIF-L displayed excellent synergistic characteristics with a large surface area, excellent conductivity, plentiful active sites, and high electrocatalytic properties, thus endowing it with high sensitivity for BM determination. The experimental parameters, such as solution pH, material volume, and accumulation time, were optimized. Under optimal conditions, the BM sensor showed a wide linear range (0.009-10.0 µM) and low-limit detection (3.0 nM). Moreover, the sensor displayed excellent stability, repeatability, and reproducibility, and good anti-interference capability. The method was successfully applied to detect BM in real-world samples.

Suppression of Arbuscular Mycorrhizal Fungi Aggravates the Negative Interactive Effects of Warming and Nitrogen Addition on Soil Bacterial and Fungal Diversity and Community Composition.

We examined the impacts of warming, nitrogen (N) addition, and suppression of arbuscular mycorrhizal fungi (AMF) on soil bacterial and fungal richness and community composition in a field experiment. AMF root colonization and the concentration of an AMF-specific phospholipid fatty acid (PLFA) were significantly reduced after the application of the fungicide benomyl as a soil drench. Warming and N addition had no independent effects but interactively decreased soil fungal richness, while warming, N addition, and AMF suppression together reduced soil bacterial richness. Soil bacterial and fungal species diversity was lower with AMF suppression, indicating that AMF suppression has a negative effect on microbial diversity. Warming and N addition decreased the net loss of plant species and the plant species richness, respectively. AMF suppression reduced plant species richness and the net gain of plant species but enhanced the net loss of plant species. Structural equation modeling (SEM) demonstrated that the soil bacterial community responded to the increased soil temperature (ST) induced by warming and the increased soil available N (AN) induced by N addition through changes in AMF colonization and plant species richness; ST directly affected the bacterial community, but AN affected both the soil bacterial and fungal communities via AMF colonization. In addition, higher mycorrhizal colonization increased the plant species richness by increasing the net gains in plant species under warming and N addition. IMPORTANCE AMF can influence the composition and diversity of plant communities. Previous studies have shown that climate warming and N deposition reduce the effectiveness of AMF. However, how AMF affect soil bacterial and fungal communities under these global change drivers is still poorly understood. A 4-year field study revealed that AMF suppression decreased bacterial and fungal diversity irrespective of warming or N addition, while AMF suppression interacted with warming or N addition to reduce bacterial and fungal richness. In addition, bacterial and fungal community compositions were determined by mycorrhizal colonization, which was regulated by soil AN and ST. These results suggest that AMF suppression can aggravate the severe losses to native soil microbial diversity and functioning caused by global changes; thus, AMF play a vital role in maintaining belowground ecosystem stability in the future.

Transcriptome analysis of Apis mellifera under benomyl stress to discriminate the gene expression in response to development and immune systems.

The health and safety of the honeybees are seriously threatened due to the abuse of chemical pesticides in modern agriculture and apiculture. In this study, the RNA Seq approach was used to assess the effects of the honeybees treated with benomyl. The results showed that there were a total of 11,902 differentially expressed genes (DEGs). Among them, 5,759 DEGs were up-regulated and involved in the functions of immunity, detoxification, biological metabolism, and regulation. The DEGs were clustered in the GO terms of epidermal structure and response to external stimuli, and most of the DEGs were enriched in 15 pathways, such as light conduction, MAPK, calcium ion pathway, and so on. Moreover, the pathway of the toll signal transduction was activated. The data investigated that the expression of functional genes involved in the growth, development, foraging, and immunity of honeybees were significantly affected by benomyl stress, which would seriously threaten the health of the honeybees. This study provided a theoretical basis for revealing the response mechanism of honeybees to pesticides stress.

Temporal and spatial analysis of benomyl/carbendazim in water and its possible impact on Nile tilapia (Oreochromis niloticus) from Tenango dam, Puebla, Mexico.

Activities like agriculture contribute to the pollution of aquatic systems by fungicides, such as benomyl/carbendazim. This chemical inhibits the activity of acetylcholinesterase (AChE), having teratogenic, oncogenic, reproductive, and hepatic effects on aquatic and soil organisms. This paper presents the results of a study conducted in the Tenango dam, Mexico, aimed at detecting and determining the spatial and temporal variability of benomyl/carbendazim fungicide in the dam's water and its possible impact on Nile tilapia (Oreochromis niloticus), farmed and commercialized in the site. Five site visits were made during 2015. Benomyl/carbendazim was quantified at 34 georeferenced stations. Thirty O. niloticus specimens were collected per visit. The quality of water and O. niloticus specimens was evaluated according to the Mexican standards. The fungicide concentrations in the O. niloticus muscle and the AChE activity were measured. Seasonal and spatial variations of benomyl/carbendazim were determined using geostatistical methods (ordinary kriging [OK] and universal kriging [UK]). Geostatistical analyses demonstrated that agriculture contributes to the increased amounts of the chemical in specific areas. Even though the fungicide levels in water varied over time, they did not represent a risk to O. niloticus according to the current standards. The specimens met the quality criteria for their commercialization; however, they had low weights and small sizes. The benomyl/carbendazim concentration in the muscle increased with the size and exhibited a negative correlation with the AChE activity, thus indicating a potential harmful effect.

Composition and function of soil fungal community during the establishment of Quercus acutissima (Carruth.) seedlings in a Cd-contaminated soil.

This study was designed to explore the functions of soil fungal communities in the Cd tolerance of Q. acutissima seedling. Three Cd levels of 15, 30, and 40 mg kg-1 were set up using the soils collected from Q. acutissima forests. The benomyl was applied to inhibit the fungal communities in the soil. Following a 100-day pot cultivation of Q. acutissima seedlings, the plant growth, Cd content, N uptake, and fungal communities were evaluated. The results showed that the root dry weights were significantly reduced after the benomyl addition at the Cd concentrations of 30 and 40 mg kg-1. Root fungi colonization was enhanced under higher Cd concentrations when soil fungi are present (without the benomyl treatment). The fungi associated with root increased the Cd accumulation in the roots while decreased the Cd transfer to the shoot at 40 mg Cd kg-1. The 15N enrichment in root tip was positively correlated with enzyme activities of soil catalase and urease. And the activities of acid phosphatase, catalase, and urease were inhibited at each Cd level. The abundance of the dominant fungal genus differed in their response to Cd contamination. The ectomycorrhizal fungi of Tomentella and Cortinarius were identified under the higher Cd levels (40 mg kg-1). Our results implied Tomentella and Cortinarius could be applied to enhance the capacity of Quercus acutissima in the bioremediation of Cd polluted soil.

Molecularly imprinted polymers for the detection of benomyl residues in water and soil samples.

Benomyl is a benzimidazol fungicide used against various crop pathogens. Although banned in many countries, it is still widely used worldwide and is listed in different monitoring programs among the substances to be monitored to assess human exposure to pesticide residues. The assessment of benomyl is mainly based on the analysis of the residues of its most important metabolite, carbendazim. Existing methods often lack of selectivity and display a limited performance because of the presence of co-extracted compounds. Molecularly imprinted polymers (MIPs) offer an alternative methodology, adsorbing preferentially those target molecules for which the polymers are specifically prepared. In this study, we optimized the synthesis of a polymer imprinted with benomyl. Tests of specificity recognition showed a good performance for carbendazim compared with other similar pesticides. The mean recovery of benomyl (measured as carbendazim) from water samples was estimated to be 90% for MIPs while with real soil samples collected in Morocco the recovery efficiency was 62%. Preliminary tests also suggest that this MIP can implement traditional SPE techniques for assessing benomyl residual concentrations in environmental samples.

Npa3/ScGpn1 carboxy-terminal tail is dispensable for cell viability and RNA polymerase II nuclear targeting but critical for microtubule stability and function.

Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.

Defoliation and arbuscular mycorrhizal fungi shape plant communities in overgrazed semiarid grasslands.

Overgrazing substantially contributes to global grassland degradation by decreasing plant community productivity and diversity through trampling, defoliation, and removal of nutrients. Arbuscular mycorrhizal (AM) fungi also play a critical role in plant community diversity, composition, and primary productivity, maintaining ecosystem functions. However, interactions between grazing disturbances, such as trampling and defoliation, and AM fungi in grassland communities are not well known. We examined influences of trampling, defoliation, and AM fungi on semiarid grassland plant community composition for 3 yr, by comparing all combinations of these factors. Benomyl fungicide was applied to reduce AM fungal abundance. Overgrazing typically resulted in reduced dominance of Stipa Krylovii, contributing to degradation of typical steppe grasslands. Our results indicated trampling generally had little effect on plant community composition, unless combined with defoliation or AM fungal suppression. Defoliation was the main component of grazing that promoted dominance of Potentilla acaulis over Stipa krylovii and Artemisia frigida, presumably by alleviating light limitation. In non-defoliated plots, AM fungi promoted A. frigida, with a concomitant reduction in S. krylovii growth compared to corresponding AM suppressed plots. Our results indicate AM fungi and defoliation jointly suppress S. krylovii biomass; however, prolonged defoliation weakens mycorrhizal influence on plant community composition. These findings give new insight into dominant plant species shifts in degraded semiarid grasslands.

The genetic architecture in Saccharomyces cerevisiae that contributes to variation in drug response to the antifungals benomyl and ketoconazole.

The genetic basis of variation in drug response was investigated in individual Saccharomyces cerevisiae strains that exhibited different susceptibility to two antifungal agents: benomyl and ketoconazole. Following dose-response screening of 25 strains, 4 were selected on the basis of resistance or sensitivity relative to the standard laboratory strain BY. UWOPS87-2421 and L-1374 were respectively resistant and sensitive to benomyl; DBVPG6044 and Y12 were respectively resistant and sensitive to ketoconazole. We used advanced intercross lines and next generation sequencing-bulk segregant analysis to characterise the quantitative trait loci (QTL) underpinning drug responses after drug selection. Drug response was controlled by multiple QTL, ranging from a minimum of 5 to a maximum of 60 loci, almost all of which were not the primary drug target. For each drug, the resistant and the sensitive strain exhibited a number of shared loci, but also had strain-specific QTL. In our analysis, it was possible to estimate genetic effect of QTL, and a number of those shared between resistant and sensitive strains exhibited variable effect on the response phenotype. Thus, drug responses arise as a result of different genetic architectures, depending on the genetic background of the individual strain in question.

Development and validation of benomyl birdseed agar for the isolation of Cryptococcus neoformans and Cryptococcus gattii from environmental samples.

One of the difficulties of isolating Cryptococcus neoformans and Cryptococcus gattii from environmental samples is the abundant overgrowth of other yeast and mold species that occurs on the plates. Here we report the application of benomyl to Guizotia abyssinica seed extract growth medium to improve the isolation of C. neoformans and C. gattii from environmental samples. We validated this medium by recovering C. neoformans and C. gattii from convenience soils and swabs from a region of the United States where these yeasts are endemic.

Influence of the agrochemical benomyl on Cryptococcus gattii-plant interaction in vitro and in vivo.

Cryptococcus gattii, an environmental fungus, is one of the agents of cryptococcosis. The influence of agrochemicals on fungal resistance to antifungals is widely discussed. However, the effects of benomyl (BEN) on fungal interaction with different hosts is still to be understood. Here we studied the influence of adaptation to BEN in the interaction with a plant model, phagocytes and with Tenebrio molitor. First, the strain C. gattii L24/01 non-adapted (NA), adapted (A) to BEN, and adapted with further culture on drug-free media (10p) interact with Nicotiana benthamiana, with a peak in the yeast burden on the 7th day post-inoculation. C. gattii L24/01 A and 10p provided lower fungal burden, but these strains increased cell diameter and capsular thickness after the interaction, together with decreased fungal growth. The strains NA and A showed reduced ergosterol levels, while 10p exhibited increased activity of laccase and urease. L24/01 A recovered from N. benthamiana was less engulfed by murine macrophages, with lower production of reactive oxygen species. This phenotype was accompanied by increased ability of this strain to grow inside macrophages. Otherwise, L24/01 A showed reduced virulence in the T. molitor larvae model. Here, we demonstrate that the exposure to BEN, and interaction with plants interfere in the morphophysiology and virulence of the C. gattii.

Ti3C2Tx/laser-induced graphene-based micro-droplet electrochemical sensing platform for rapid and sensitive detection of benomyl.

The design and fabrication of high-performance electrode devices are highly important for the practical application of electrochemical sensors. In this study, flexible three-dimensional porous graphene electrode devices were first facilely fabricated using common laser ablation technique at room temperature. After then, hydrophilic two-dimensional MXene (Ti3C2Tx) nanosheet was decorated on the surface of the laser-induced graphene (LIG), resulting in disposable Ti3C2Tx/LIG electrode devices. After introducing Ti3C2Tx nanosheet, the electrochemical active area, electron transfer ability of LIG electrode device and its adsorption efficiency toward organic pesticide benomyl was significantly boosted. As a result, the fabricated Ti3C2Tx/LIG electrode device exhibited significantly enhanced electrocatalytic activity toward benomyl oxidation. Based on this, a novel and ultra-sensitive electrochemical platform for micro-droplet detection of benomyl was achieved in the range of 10 nM-6000 nM with detection sensitivity of 169.9 µA µM-1 cm-2 and detection limit of 5.8 nM. Considering the low-cost Ti3C2Tx/LIG electrode devices are rarely used for electrochemical analysis, we believed this research work will contribute to exploring the broader application of MXene/LIG electrode devices in the field of electrochemical sensing.

Benomyl-induced development and cardiac toxicity in zebrafish embryos.

Benomyl is a highly effective broad-spectrum fungicide widely used worldwide to control vegetable, fruit, and oil crop diseases. However, the mechanism of its toxicity to aquatic organisms and humans remains unknown. In this study, zebrafish were used to determine the toxicity of benomyl. It was found to be highly toxic, with a 72-h post-fertilization (hpf) lethal concentration 50 (LC50) of 1.454 mg/L. Benomyl induced severe developmental toxicity, including shorter body length, slower heart rate, and a reduced yolk absorption rate. Benomyl also increased oxidative stress in zebrafish, especially in the heart and head, as well as increasing malondialdehyde (MDA) content and decreasing catalase (CAT) and superoxide dismutase (SOD) activities. This indicates that benomyl induced reactive oxygen species (ROS) production and cell membrane peroxidation in vivo. Acridine orange (AO) staining and apoptosis factor detection further indicated that benomyl induced apoptosis in zebrafish. Overall, these findings demonstrate that benomyl disrupts cellular homeostasis by activating oxidative stress in zebrafish, resulting in an imbalance of cardiac development-related gene expression and apoptosis, which causes severe developmental toxicity and cardiac dysfunction. This study evaluated the in vivo toxicity of benomyl, which is a potential threat to aquatic organisms and humans. Possible toxicity mechanisms are explored, providing a valuable reference for the safe use of benomyl.

Investigating and assessing the risk of remaining toxins in cucumbers grown in fields and greenhouses in Mazandaran Province.

To address concerns regarding the potential health risks associated with residual chemicals in food products, this study aimed to assess the concentrations of residual Benomyl, Malathion, and Diazinon in cucumbers and evaluate the associated health risks for consumers. This descriptive study involved the collection of 100 cucumber samples from both field and greenhouse cultivation in Mazandaran Province. These samples were subsequently sent to the laboratory for analysis. Following sample preparation and digestion, we determined the levels of residual Malathion, Benomyl, and Diazinon using HPLC. The results showed an average residual Malathion concentration of 2.1 ± 0.04 mg/kg in field-grown cucumbers and 2.04 ± 1.5 mg/kg in greenhouse-cultivated cucumbers. Meanwhile, the average residual Diazinon concentration was 5.1 ± 0.2 mg/kg in field samples and 4.99 ± 3.23 mg/kg in greenhouse samples. The average concentrations of residual Benomyl were found to be 0.94 ± 0.65 mg/kg in field-grown cucumbers and 0.39 ± 0.2 mg/kg in greenhouse-cultivated cucumbers. Furthermore, a health risk assessment model was employed to evaluate the cumulative risk of these residual pesticides in cucumbers. The analysis revealed that residual Benomyl levels ranged from 0 to 24.33 mg/kg, while Malathion ranged from 0 to 9.25 mg/kg, and Diazinon ranged from 0 to 6.8 mg/kg. Notably, in some areas, the average concentration of all three pesticides exceeded the guidelines set by the WHO and the European Union. Additionally, the cumulative risk assessment (represented by MOET value of 2655) indicated that the combined presence of residual Malathion, Benomyl, and Diazinon in field-cultivated cucumbers posed a health risk. Based on current per capita consumption rates, the study's health risk index results raised concerns about the safety of consuming these residual byproducts. Given the growing use of chemicals in agricultural and horticultural practices, it is imperative to monitor residual pesticides to assess dietary intake and ensure consumer safety in food production.

Effect of benomyl-mediated mycorrhizal association on the salinity tolerance of male and monoecious mulberry clones.

Mulberry is an economically important crop for sericulture in China. Mulberry plantations are shifting inland, where they face high salinity. Arbuscular mycorrhizal fungi (AMF) reportedly enhance mulberry's tolerance to salinity. Here, we assessed if additional adaptive advantages against salinity are provided by sex differences beyond those provided by mycorrhizal symbiosis. In a pot experiment, male and monoecious plants were exposed to three salinity regimes (0, 50, and 200 mM NaCl) and two mycorrhiza-suppressed conditions (with or without benomyl application) for more than 16 months. We noticed that salinity alone significantly decreased the mycorrhizal colonization rate, salinity tolerance, K+ concentrations, and the ionic ratios of all plants. Mycorrhizal association mildly ameliorated the salt-induced detrimental effects, especially for monoecious plants, and sex-specific responses were observed. Meanwhile, both sexes had adopted different strategies to enhance their salinity resistance. Briefly, mycorrhizal monoecious plants exhibited a higher net photosynthetic rate and lower translocation of Na+ from root to shoot compared with mycorrhizal males under saline conditions. Their salt tolerance was probably due to the Ca2+/Na+ in roots. In comparison, male plants exhibited lower Na+ acquisition, more Na+ translocated from root to shoot, higher root biomass allocation, and higher N concentrations under harsh saline conditions, and their salt tolerance was mainly related to the K+/Na+ in their shoots. In conclusion, our results highlight that AMF could be a promising candidate for improving plant performance under highest salinity, especially for monoecious plants. Cultivators must be mindful of applying fungicides, such as benomyl, in saline areas.

Detection of Colletotrichum spp. Resistant to Benomyl by Using Molecular Techniques.

Colletotrichum species is known as the major causal pathogen of red pepper anthracnose in Korea and various groups of fungicides are registered for the management of the disease. However, the consistent use of fungicides has resulted in the development of resistance in many red pepper-growing areas of Korea. Effective management of the occurrence of fungicide resistance depends on constant monitoring and early detection. Thus, in this study, various methods such as agar dilution method (ADM), gene sequencing, allele-specific polymerase chain reaction (PCR), and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were applied for the detection of benzimidazole resistance among 24 isolates of Colletotrichum acutatum s. lat. and Colletotrichum gloeosporioides s. lat. The result of the ADM showed that C. gloeosporioides s. lat. was classified into sensitive and resistant isolates to benomyl while C. acutatum s. lat. was insensitive at ≥1 µg/ml of benomyl. The sequence analysis of the ß-tubulin gene showed the presence of a single nucleotide mutation at the 198th amino acid position of five isolates (16CACY14, 16CAYY19, 15HN5, 15KJ1, and 16CAYY7) of C. gloeosporioides s. lat. Allele-specific PCR and PCR-RFLP were used to detect point mutation at 198th amino acid position and this was done within a day unlike ADM which usually takes more than one week and thus saving time and resources that are essential in the fungicide resistance management in the field. Therefore, the molecular techniques established in this study can warrant early detection of benzimidazole fungicide resistance for the adoption of management strategies that can prevent yield losses among farmers.

Chemical reactivity, molecular electrostatic potential and in-silico analysis on benzimidazole fungicide benomyl.

We are reporting theoretical concepts and biological activity of benomyl using different techniques. The molecular orbital contributions are studied by using Total Density of States (TDOS) analysis. The chemical reactivity of the molecule have been determined with the help of global reactivity descriptors. Molecular electrostatic potential is calculated by the density functional method and predicts the most reactive part in the molecule. In-silico molecular analysis is conducted for Benomyl compound.