Comparative fitness of Monilinia fructicola isolates with multiple fungicide-resistant phenotypes.

This study characterized 52 isolates of Monilinia fructicola from peach and nectarine orchards for their multi-resistance patterns to thiophanate-methyl (TF), tebuconazole (TEB), and azoxystrobin (AZO) using in vitro sensitivity assays and molecular analysis. The radial growth of M. fructicola isolates was measured on media amended with a single discriminatory dose of 1 µg/ml for TF and AZO and 0.3 µg/ml for TEB. Cyt b, CYP51, and ß-tubulin were tested for point mutations that confer resistance to quinone outside inhibitors (QoIs), demethylation inhibitors (DMIs), and methyl benzimidazole carbamates (MBCs), respectively. Eight phenotypes were identified including isolates with single, double, and triple in vitro resistance to QoI, MBC, and DMI fungicides. All resistant phenotypes to TF and TEB presented the H6Y mutation in ß-tubulin and the G641S mutation in CYP51. None of the point mutations typically linked to QoI resistance were present in the Monilinia isolates examined. Moreover, fitness of the M. fructicola phenotypes was examined in vitro and detached fruit assays. Phenotypes with single-resistance displayed equal fitness in in vitro and fruit assays compared to the wild-type. In contrast, the dual and triple-resistance phenotypes suffered fitness penalties based on osmotic sensitivity and aggressiveness on peach fruit. In this study, multiple resistance to MBC, DMI, and QoI fungicide groups was confirmed in M. fructicola. Results suggest that Monilinia populations with multiple resistance phenotypes are likely to be less competitive in the field than those with single resistance, thereby impeding their establishment over time and facilitating disease management.

Fungicide Sensitivity of Fusarium oxysporum f. sp. lentis and Fusarium acuminatum Affecting Lentil in the Northern Great Plains.

Fusarium oxysporum f. sp. lentis and F. acuminatum cause wilting and root rot in pulse crops including lentil. Fungicide seed treatments are widely used, but information about Fusarium spp. sensitivity in lentils is limited. Here, 30 F. oxysporum f. sp. lentis and 30 F. acuminatum isolates from Montana, southern Canada, North Dakota, and Washington were identified, tested for pathogenicity, and assayed for in vitro sensitivity to pyraclostrobin, prothioconazole, ipconazole, and thiophanate-methyl. F. oxysporum f. sp. lentis and F. acuminatum differed in their sensitivity to all fungicides. No resistant isolates were identified, but F. oxysporum f. sp. lentis had lower EC50 values in pyraclostrobin (averaging 0.47 µg a.i./ml) than F. acuminatum (averaging 0.89 µg a.i./ml) for mycelia assays. Both species had lower EC50 values in prothioconazole, averaging EC50 0.23 in F. oxysporum f. sp. lentis and 0.53 µg a.i./ml in F. acuminatum. F. oxysporum f. sp. lentis isolates had the lowest EC50 values on ipconazole compared to F. acuminatum (0.78 and 1.49 µg a.i./ml). The pathogens were least sensitive to thiophanate-methyl (1.74 µg a.i./ml for F. oxysporum f. sp. lentis and 1.91 µg a.i./ml for F. acuminatum). Overall sensitivity to the fungicides was higher in F. oxysporum f. sp. lentis than F. acuminatum. This study provides reference EC50 values while pointing to the possibility of differential fungicide efficacies on Fusarium spp. This will be helpful to monitor shifts in sensitivity of Fusarium spp. and devise robust root rot/wilt management approaches.

MOF-based Ag NPs/Co3O4 nanozyme for colorimetric detection of thiophanate-methyl based on analyte-enhanced sensing mechanism.

Silver nanoparticles supported on metal-organic framework (ZIF-67)-derived Co3O4 nanostructures (Ag NPs/Co3O4) were synthesized via a facile in situ reduction strategy. The resulting materials exhibited pH-switchable peroxidase/catalase-like catalytic activity. Ag NP doping greatly enhanced the catalytic activity of Ag NPs/Co3O4 towards 3,3',5,5'-tetramethylbenzidine (TMB) oxidation and H2O2 decomposition which were 59 times (A652 of oxTMB) and 3 times (A240 of H2O2) higher than that of ZIF-67, respectively. Excitingly, thiophanate-methyl (TM) further enhanced the peroxidase-like activity of Ag NPs/Co3O4 nanozyme due to the formation of Ag(I) species in TM-Ag NPs/Co3O4 and generation of more radicals resulting from strong interaction between Ag NPs and TM. The TM-Ag NPs/Co3O4 nanozyme exhibited lower Km and higher Vmax values towards H2O2 when compared with Ag NPs/Co3O4 nanozyme. A simple, bioelement-free colorimetric TM detection method based on Ag NPs/Co3O4 nanozyme via analyte-enhanced sensing strategy was successfully established with high sensitivity and selectivity. Our study demonstrated that hybrid noble metal NPs/MOF-based nanozyme can be a class of promising artificial nanozyme in environmental and food safety applications.

Low frequency of resistance to thiophanate-methyl in Monilinia fructicola populations from southeastern United States peach orchards.

Methyl benzimidazole carbamate (MBC) fungicides were once widely used for brown rot (Monilinia fructicola) control of peach (Prunus persica (L.) Batsch) in the southeastern US, but their use was substantially reduced and often eliminated due to widespread resistance. In this study, 233 M. fructicola isolates were collected from major peach production areas in Alabama, Georgia, and South Carolina, and sensitivity to thiophanate-methyl was examined. Isolates were also collected from one organic and two experimental peach orchards. A discriminatory dose of 1 µg/ml was used to distinguish sensitive (S) and moderately sensitive (S-LR) isolates from low resistant phenotypes, while 50 and 500 µg/ml thiophanate-methyl concentrations were used to determine high resistant (HR) phenotypes. Sequence analyses were performed to identify mutations in the ß-tubulin target gene and detached fruit assays were performed to determine the efficacy of a commercial product against isolates representing each phenotype. Results indicated 55.7%, 63.5%, and 75.9% of isolates from Alabama, Georgia, and South Carolina, respectively, were S to thiophanate-methyl; 44.3%, 36.5%, and 21.4% were S-LR; no isolates were LR; and only 3 isolates (1.3%) from South Carolina were HR. No mutations in S or S-LR isolates were found, but HR isolates revealed the E198A mutation, an amino acid change of glutamic acid to alanine conferring high resistance. The high label rate of a commercial product containing thiophanate-methyl controlled brown rot caused by S and S-LR isolates in detached fruit studies but was ineffective against HR isolates. The combinations of thiophanate-methyl with azoxystrobin or isofetamid, when mixed together and applied in an experimental orchard 14 days preharvest, significantly reduced brown rot incidence on pre and postharvest commercially ripe fruit and efficacy was comparable to that of a grower standard fungicide. These results indicate that thiophanate-methyl may again be useful to peach growers in the southeastern US for brown rot and fungicide resistance management.

Fungicide Sensitivity of Sclerotinia sclerotiorum from U.S. Soybean and Dry Bean, Compared to Different Regions and Climates.

Fungicide use is integral to reduce yield loss from Sclerotinia sclerotiorum on dry bean and soybean. Increasing fungicide use against this fungus may lead to resistance to the most common fungicides. Resistance has been reported in Brazil (Glycine max) and China (Brassica napus subsp. napus), however, few studies have investigated fungicide sensitivity of S. sclerotiorum in the United States. This work was conducted to determine if there was a difference in fungicide sensitivity of S. sclerotiorum isolates in the United States from: (i) dry bean versus soybean and (ii) fields with different frequencies of fungicide application. We further hypothesized that isolates with fungicide applications of a single active ingredient from tropical Brazil and subtropical Mexico were less sensitive than temperate U.S. isolates due to different management practices and climates. The EC50(D) fungicide sensitivity of 512 S. sclerotiorum isolates from the United States (443), Brazil (36), and Mexico (33) was determined using a discriminatory concentration (DC) previously identified for tetraconazole (2.0 ppm; EC50(D) range of 0.197 to 2.27 ppm), boscalid (0.2; 0.042 to 0.222), picoxystrobin (0.01; 0.006 to 0.027), and thiophanate-methyl, which had a qualitative DC of 10 ppm. Among the 10 least sensitive isolates to boscalid and picoxystrobin, 2 presented mutations known to confer resistance in the SdhB (qualitative) and SdhC (quantitative) genes; however, no strong resistance was found. This study established novel DCs that can be used for further resistance monitoring and baseline sensitivity of S. sclerotiorum to tetraconazole worldwide plus baseline sensitivity to boscalid in the United States.

Molecular Diagnosis of Thiophanate-Methyl-Resistant Strains of Fusarium fujikuroi in Japan.

Fusarium fujikuroi is the pathogen of rice bakanae disease and is subclassified into gibberellin and fumonisin groups (G and F groups). Thiophanate-methyl (TM), a benzimidazole fungicide, has been used extensively to control F. fujikuroi. Previous investigation showed that F-group strains are TM sensitive (TMS), whereas most G-group strains are TM resistant (TMR) in Japan. The minimum inhibitory concentration in TMS strains was 1 to 10 µg ml-1, whereas that in TMR strains was >100 µg ml-1. E198K and F200Y mutations in ß2-tubulin were detected in TMR strains. A loop-mediated isothermal amplification-fluorescent loop primer method was developed for diagnosis of these mutations and applied to 37 TMR strains and 56 TMS strains. The results indicated that 100% of TMR strains were identified as having either the E198K mutation (41%) or the F200Y mutation (59%), whereas none of the TMS strains tested showed either mutation. We found one remarkable TMR strain in the F group that had an F200Y mutation. These results suggest that E198K and F200Y mutations in ß2-tubulin contribute to TM resistance in F. fujikuroi.

Fungicide Sensitivity of Colletotrichum Species Causing Bitter Rot of Apple in the Mid-Atlantic U.S.A.

Apple growers in the Mid-Atlantic region of the U.S.A. have reported increased losses to bitter rot of apple. We tested the hypothesis that this increase is because the Colletotrichum population has developed resistance to commonly used single-mode-of-action (single-MoA) fungicides. We screened 220 Colletotrichum isolates obtained from 38 apple orchards in the Mid-Atlantic region for resistance to 11 fungicides in Fungicide Resistance Action Committee (FRAC) groups 1, 7, 9, 11, 12, and 29. Eleven (5%) of these isolates were resistant to FRAC group 1 with confirmed ß-tubulin E198A mutations, and two (<1%) were also resistant to FRAC group 11 with confirmed cytochrome-b G143A mutations. Such low frequencies of resistant isolates indicate that fungicide resistance is unlikely to be the cause of any regional increase in bitter rot. A subsample of isolates was subsequently tested in vitro for sensitivity to every single-MoA fungicide registered for apple in the Mid-Atlantic U.S.A. (22 fungicides; FRAC groups 1, 3, 7, 9, 11, 12, and 29), and 13 fungicides were tested in field trials. These fungicides varied widely in efficacy both within and between FRAC groups. Comparisons of results from our in vitro tests with results from our field trials and other field trials conducted across the eastern U.S.A. suggested that EC25 values (concentrations that reduce growth by 25%) are better predictors of fungicide efficacy in normal field conditions than EC50 values. We present these results as a guideline for choosing single-MoA fungicides for bitter rot control in the Mid-Atlantic U.S.A.

Residue analysis and kinetics modeling of thiophanate-methyl, carbendazim, tebuconazole and pyraclostrobin in apple tree bark using QuEChERS/HPLC-VWD.

Winter is the key period for the control of apple diseases, and fungicides are needed to protect the trunk or main branches. Fungicide residue in apple tree bark is an important basis for the action of the pesticide, but there are no reports on analytical methods or dissipation patterns. In this work, thiophanate-methyl, carbendazim, tebuconazole and pyraclostrobin were selected as typical fungicides and a new QuEChERS-HPLC-VWD(QuEChERS extraction followed by high-performance liquid chromatography detection with a variable wavelength detector) analytical method was developed to estimate their residue kinetics in apple tree bark during the winter months. In the pretreatment step, the sorbent for the clean-up of extracts was optimized as 60 mg/ml primary secondary amine and a gradient-elution model followed by a variable wavelength detection was developed for instrumental analysis. Then this method was validated and applied to the analysis of apple tree bark samples with the linearity range of 0.010-50.00 mg/L, quantification limit range of 0.028-0.080 mg/kg and recovery range of 86.1-101.4%. The dissipation kinetics of thiophanate-methyl and pyraclostrobin could be described by the first-order and two-phase kinetics models, respectively. For carbendazim and tebuconazole, two new models were developed to describe their residue kinetics.

Analysis of the dissipation kinetics of thiophanate-methyl and its metabolite carbendazim in apple leaves using a modified QuEChERS-UPLC-MS/MS method.

As one of the main fungicides for the apple leaf disease control, thiophanate-methyl (TM) mainly exerts its fungicidal activity in the form of its metabolite carbendazim (MBC), whose dissipation kinetics is very distinct from that of its parent but has been paid little attention. The aim of this work was to investigate the dissipation kinetics of TM and its active metabolite MBC in apple leaves using a modified QuEChERS-UPLC-MS/MS method. The results showed that TM and MBC could be quickly extracted by this modified QuEChERS procedure with recoveries of 81.7-96.5%. The method linearity was in the range of 0.01-50.0 mg kg-1 with the quantification limit of 0.01 mg kg-1 . Then this method was applied to the analysis of fungicide dissipation kinetics in apple leaves. The results showed that the dissipation kinetics of TM for the test in 3 months can be described by a first-order kinetics model with a DT50 (dissipation half-life) range of 5.23-6.03 days and the kinetics for MBC can be described by a first-order absorption-dissipation model with the Tmax (time needed to reach peak concentration) range of 4.78-7.09 days. These models can scientifically describe the behavior of TM and MBC in apple leaves, which provides necessary data for scientific application.

Immunomodulatory effect of mancozeb, chlorothalonil, and thiophanate methyl pesticides on macrophage cells.

Mancozeb (MZ), chlorothalonil (CT), and thiophanate methyl (TM) are pesticides commonly used in agriculture due to their efficacy, low acute toxicity to mammals, and short environmental persistence. Although the toxic effects of these pesticides have been previously reported, studies regarding their influence on the immune system are limited. As such, this study focused on the immunomodulatory effect of MZ, CT, and TM pesticides on macrophage cells. RAW 264.7 cells were exposed to a range of concentrations (0.1-100 µg/mL) of these pesticides. CT exposure promoted an increase in reactive oxygen species (ROS) and nitric oxide (NO) levels. The MTT and ds-DNA assay results demonstrated that MZ, CT, and TM exposure induced macrophage proliferation. Moreover, MZ, CT, and TM promoted cell cycle arrest at S phase, strongly suggesting macrophage proliferation. The levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, and IFN-γ) and caspases (caspase 1, 3, and 8) in macrophages exposed to MZ, CT, and TM pesticides increased, whereas the anti-inflammatory cytokine levels decreased. These results suggest that MZ, CT, and TM exert an immunomodulatory effect on the immune system, inducing macrophage activation and enhancing the inflammatory response.

Itraconazole and thiophanate-methyl fail to clear tadpoles naturally infected with the hypervirulent lineage of Batrachochytrium dendrobatidis.

The emerging infectious disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis, is a major driver pushing many amphibian species to the brink of extinction. Substantial efforts to develop effective protocols that use antifungal drugs have had notable success. Here, we used the antifungal agents itraconazole and thiophanate-methyl, singly and in combination, in an attempt to treat common midwife toad Alytes obstetricans larvae naturally infected with the globalized hypervirulent lineage of B. dendrobatidis. Despite the successful use of itraconazole in a closely related species (A. muletensis), our results show that these antifungal treatments are not always effective and that full clearance of animals cannot be assumed following treatment.

Thiophanate-methyl resistance in Sclerotinia homoeocarpa from golf courses in China.

Sclerotinia homoeocarpa causes dollar spot disease on many turfgrass species and is a significant problem worldwide. Thiophanate-methyl (TM), a methyl benzimidazole carbamate (MBC) fungicide, has been used for over forty years to manage dollar spot. Here we describe genetic mutations linked to three distinct TM fungicide resistance phenotypes: sensitive (S), moderately resistant (MR) and highly resistant (HR). These were established using multiple doses of TM, compared to previous studies using single discriminatory doses. In total, 19 S, 3 MR and 22 HR isolates were detected. Analysis of the ß-tubulin gene revealed the MR isolates had a point mutation from T to A at codon 200 changing phenylalanine (TTC) to tyrosine (TAC). Twenty HR isolates had a mutation at codon 198 changing glutamic acid (GAG) to alanine (GCG) and two HR isolates had a mutation at codon 198 changing glutamic acid (GAG) to lysine (AAG). Allele-specific PCR assays were developed for rapid detection of these mutations in isolates of S. homoeocarpa. In addition, our results suggest a two-dose system for in vitro screening provides useful information for monitoring the development of resistance.

Methyl thiophanate-induced toxicity in liver and kidney of adult rats: a biochemical, molecular and histopathological approach.

The aim of this study was to elucidate the redox effects of Thiophanate methyl (MT) in the rat liver and kidney. Our results showed, after 3 days of MT injection (700 mg/kg), an increase in malondialdehyde (MDA), hydrogen peroxide and advanced oxidation protein products levels. Glutathione peroxidase and superoxide dismutase activities were also remarkably increased in the liver but decrease in the kidney. Glutathione and vitamin C values were significantly reduced. The changes in biochemical parameters were substantiated by histological and molecular data. A smear without ladder formation on agarose gel was shown, indicating random DNA degradation in the liver and the kidney of MT treated rats. The increase in cyclooxygenase-2 gene expression, marker of inflammation, and an increase in genes expression of glutathione peroxidase and superoxide dismutase in liver and their decrease in the kidney were also occurred after MT exposure. These data confirmed the pro-oxidant and genotoxic effects of this fungicide.

Behavior of Thiophanate Methyl and Propiconazole in Grape and Mango Fruits Under the Egyptian Field Conditions.

This research aims at determining residues of thiophanate methyl and propiconazole in grape and mango fruits as an indication for their persistence in this environmental compartment. Fruit extracts were analyzed for thiophanate methyl using High Performance Liquid Chromatography and using Gas Chromatography Electron Capture Detector (GC/ECD), respectively. The results indicated that propiconazole had a less environmental impact since propiconazole had shorter residue half-lives which were 1.24 and 1.19 days in grape and mango fruits, respectively, while thiophanate methyl had half-lives of 2.49 and 2.64 days in mango and grape, respectively. The degradation rates of propiconazole in grape and mango fruits did not change significantly and neither did those of thiophanate methyl. According to the maximum residue level, the pre-harvest intervals of propiconazole were set to be 3 and 7 days for grape and mango fruits, respectively, and the pre-harvest intervals for thiophanate methyl were 15 days for both grape and mango fruits. Propiconazole was generally considered to be less hazardous to humans and will leave the environment less altered because of its faster degradation than that of thiophanate methyl.

Sensitive colorimetric and fluorescence dual-mode detection of thiophanate-methyl based on spherical Fe3O4/GONRs composite nanozyme.

Pesticide detection based on nanozyme is largely limited in terms of the variety of pesticides. Herein, a spherical and well-dispersed Fe3O4/graphene oxide nanoribbons (Fe3O4/GONRs) composite nanozyme was applied to firstly develop an enzyme-free and sensitive colorimetric and fluorescence dual-mode detection of thiophanate-methyl (TM). The synthesized Fe3O4/GONRs possess excellent dual enzyme-like activities (peroxidase and catalase) and can catalyze H2O2 to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) into oxidized TMB (oxTMB). We found that Fe3O4/GONRs can adsorb TM through the synergistic effect of multiple forces, thereby inhibiting the catalytic activities of nanozyme. This inhibition can modulate the transformation of TMB to oxTMB, producing dual responses of absorbance decrease (oxTMB) and fluorescence enhancement (TMB). The limits of detection (LODs) of TM were 28.1 ng/mL (colorimetric) and 8.81 ng/mL (fluorescence), respectively. Moreover, the developed method with the recoveries of 94.8-100.8% also exhibited a good potential application in the detection of pesticides residues in water and food samples.

Synthesis of metal framework-modified carbon dots with super large stokes shift using Hami melon as a green precursor for detecting thiophanate-methyl residue in leafy vegetables.

Consuming leafy vegetables with excessive thiophanate-methyl (TM) residue poses serious risks to human health. To realize rapidly and sensitively detecting TM in leafy vegetables, we developed a fluorescent probe based on zeolitic imidazolate framework-8-modified carbon dots using Hami melon as the green precursor (HM-CDs@ZIF-8). Meanwhile, the mechanism of HM-CDs@ZIF-8 for detecting TM was investigated and explained. The results of the performance tests showed that the prepared HM-CDs@ZIF-8 exhibited high sensitivity, excellent selectivity, robust anti-interference capability, reliable reproducibility and repeatability, and long-term stability. After optimization experiments, the fluorescence intensity of HM-CDs@ZIF-8 showed a strong linear correlation with the concentration of TM (0.00171-3.4239 mol/L) with a detection limit of 2.025 µmol/L. The HM-CDs@ZIF-8 was successfully applied to determine TM in spiked leafy vegetables with satisfactory recoveries of 96-105%. The relative standard deviations were in the range of 0.26-2.55%. The sensor has a promising application for detecting TM in leafy vegetables.

Smartphone-assisted carbon dots fluorescent sensing platform for visual detection of Thiophanate-methyl in fruits and vegetables.

Trimesic acid and o-phenylenediamine (OPD) were employed as precursors to synthesize yellow-green fluorescent carbon dots (Y-G-CDs) by solvothermal synthesis for the sensitive detection of Thiophanate-methyl (TM) in real agricultural products. The Y-G-CDs probe could specifically recognize the TM primarily through π-π stacking. Moreover, the fluorescence quenching of the probe was ultimately dominated by the PET effect, based on the interaction between the abundant carboxyl groups on the surface of the Y-G-CDs and the amino group of TM. A strong linear relationship between the fluorescence quenching of the probe and TM concentration in the range of 0-10 µmol/L was observed and the limit of detection (LOD) was calculated to be 50.7 nmol/L. Compared to the interference pesticides, the Y-G-CDs probe demonstrated exceptional selectivity toward TM, with satisfactory recoveries of 96.3 % - 104.2 % in spiked food samples. The Y-G-CDs probe enables simple pretreatment, cost-effective, and on-site detection of TM in fruits and vegetables with visual detection of the TM employing a smartphone-assisted sensing platform.

Updated reasoned opinion on the toxicological properties and maximum residue levels (MRLs) for the benzimidazole substances carbendazim and thiophanate-methyl.

In compliance with Article 43 of Regulation (EC) No 396/2005, EFSA received from the European Commission in 2020 a mandate to provide its reasoned opinion on the toxicological properties and maximum residue levels (MRLs) for the benzimidazole substances carbendazim and thiophanate-methyl. Specifically, EFSA was asked to assess whether thiophanate-methyl or carbendazim has clastogenic potential and, in case clastogenic potential can be excluded, to derive toxicological reference values necessary for consumer risk assessment and assessment of maximum residue levels (MRLs). Although these active substances are no longer authorised within the European Union, MRLs were established by the Codex Alimentarius Commission (codex maximum residue limits; CXLs), and import tolerances are in place. Based on the assessment of the available data, toxicological reference values and MRL proposals were derived and a consumer risk assessment was carried out. Some information required by the regulatory framework was found to be missing and a possible acute risk to consumers was identified. Hence, the consumer risk assessment was considered indicative only and all MRL proposals derived by EFSA still require further consideration by risk managers. In October 2022, to ensure that MRLs derived by EFSA in its assessment of 2021 are safe for consumers also in view of endocrine-disrupting properties, EFSA was requested to carry out a follow-up assessment taking into account the scientific criteria for identifying endocrine disruptors (ED). Based on the outcome of the assessment, the experts agreed that the reference values are also covering the concern related to the identified hazards indicative of endocrine disruption for thiophanate-methyl. No further considerations on the impact of the ED assessment on the current reference values were needed for carbendazim since the ED criteria are not met for this substance. Therefore, the risk assessment and the MRL recommendations derived in 2021 are confirmed.

Preventive and curative effect of difenoconazole + azoxytrobin and thiophanate-methyl against lucky bamboo anthracnose disease caused by Colletotrichum dracaenophilum.

In Egypt, Dracaena sanderiana (lucky bamboo) is an ornamental plant imported from several countries. Two weeks after they arrived at the nurseries, anthracnose indications were detected on the shoots of imported D. sanderiana samples. Four Colletotrichum spp. isolates were obtained from the symptomatic lucky bamboo plants. The obtained isolates belonged to the species of C. gloeosporioides or C. dracaenophilum based on their morphological characteristics and molecular biology analyses. Pathogenicity tests reveal that C. dracaenophilum isolate 4 was found to be more pathogenic than the other isolates. The in vitro investigation was conducted with the objectives of evaluating six systemic fungicides for their inhibitory effect against C. dracaenophilum. Data reveal that, thiophanate-methyl and difenoconazole + azoxytrobin at ≥15 ppm completely inhibited the pathogen growth. Tebuconazole and flusllazole inhibited growth completely at ≥20 ppm, whereas iprodione and cyprodinil + fludioxonil had a lower effect (56.6 and 54.4% reduction, respectively) at this dose. The in vivo investigation was conducted with the objectives of evaluating the preventive and curative effects of the most effective fungicides against anthracnose disease. Lucky bamboo plants were treated with fungicide and either inoculated or not with C. dracaenophilum before being left for 25 or 60 days. On both insidiously infected and vaccinated lucky bamboo plants, the combination of difenoconazole, azoxytrobin, and thiophanate-methyl at 20 ppm greatly reduced the development of anthracnose. Tebuconazole and flusllazole were found to be phytotoxic.

Self-assembled thiophanate-methyl/star polycation complex prevents plant cell-wall penetration and fungal carbon utilization during cotton infection by Verticillium dahliae.

No effective fungicides are available for the management of Verticillium dahliae, which causes vascular wilt disease. In this study, a star polycation (SPc)-based nanodelivery system was used for the first time to develop a thiophanate-methyl (TM) nanoagent for the management of V. dahliae. SPc spontaneously assembled with TM through hydrogen bonding and Van der Waals forces to decrease the particle size of TM from 834 to 86 nm. Compared to TM alone, the SPc-loaded TM further reduced the colony diameter of V. dahliae to 1.12 and 0.64 cm, and the spore number to 1.13 × 108 and 0.72 × 108 cfu/mL at the concentrations of 3.77 and 4.71 mg/L, respectively. The TM nanoagents disturbed the expression of various crucial genes in V. dahliae, and contributed to preventing plant cell-wall degradation and carbon utilization by V. dahliae, which mainly impaired the infective interaction between pathogens and plants. TM nanoagents remarkably decreased the plant disease index and the fungal biomass in the root compared to TM alone, and its control efficacy was the best (61.20 %) among the various formulations tested in the field. Furthermore, SPc showed negligible acute toxicity toward cotton seeds. To the best of our knowledge, this study is the first to design a self-assembled nanofungicide that efficiently inhibits V. dahliae growth and protects cotton from the destructive Verticillium wilt.