Resistance risk assessment for benzovindiflupyr in Sclerotium rolfsii and transmission of resistance genes among population.

BACKGROUND: Sclerotium rolfsii is a destructive soil-borne fungal pathogen which is distributed worldwide. In previous study, the succinate dehydrogenase inhibitor (SDHI) fungicide benzovindiflupyr has been identified for its great antifungal activity against Sclerotium rolfsii. This study is aimed to investigate the resistance risk and mechanism of benzovindiflupyr in Sclerotium rolfsii. RESULTS: Eight stable benzovindiflupyr-resistant isolates were generated by fungicide adaptation. Although the obtained eight resistant isolates have a stronger pathogenicity than the parental sensitive isolate, they have a fitness penalty in the mycelial growth and sclerotia formation compared to the parental isolate. A positive cross-resistance existed in the resistant isolates between benzovindiflupyr and thifluzamide, carboxin, boscalid and isopyrazam. Three-point mutations, including SdhBN180D, SdhCQ68E and SdhDH103Y, were identified in the benzovindiflupyr-resistant isolates. However, molecular docking analysis indicated that only SdhDH103Y could influence the sensitivity of Sclerotium rolfsii to benzovindiflupyr. After mycelial co-incubation of resistant isolates and the sensitive isolate, resistance genes may be transmitted to the sensitive isolate. The in vivo efficacy of benzovindiflupyr and thifluzamide against benzovindiflupyr-resistant isolates was a little lower than that against the sensitive isolate but with no significant difference. CONCLUSION: The results suggested a low to medium resistance risk of Sclerotium rolfsii to benzovindiflupyr. However, once resistance occurs, it is possible to spread in the population of Sclerotium rolfsii. This study is helpful to understanding the risk and mechanism of resistance to benzovindiflupyr in multinucleate pathogens such as Sclerotium rolfsii. © 2024 Society of Chemical Industry.

Selection and Validation of Reference Genes for Reverse-Transcription Quantitative PCR Analysis in Sclerotium rolfsii.

Reference genes are important for the accuracy of gene expression profiles using reverse-transcription quantitative PCR (RT-qPCR). However, there are no available reference genes reported for Sclerotium rolfsii; it actually has a pretty diverse and wide host range. In this study, seven candidate reference genes (UBC, ß-TUB, 28S, 18S, PGK, EF1α and GAPDH) were validated for their expression stability in S. rolfsii under conditions of different developmental stages, populations, fungicide treatments, photoperiods and pHs. Four algorithm programs (geNorm, Normfinder, Bestkeeper and ΔCt) were used to evaluate the gene expression stability, and RefFinder was used to integrate the ranking results of four programs. Two reference genes were recommended by RefFinder for RT-qPCR normalization in S. rolfsii. The suitable reference genes were GAPDH and UBC across developmental stages, PGK and UBC across populations, GAPDH and PGK across fungicide treatments, EF1α and PGK across photoperiods, ß-TUB and EF1α across pHs and PGK and GAPDH across all samples. Four target genes (atrB, PacC, WC1 and CAT) were selected for the validation of the suitability of selected reference genes. However, using one or two reference genes in combination to normalize the expression of target genes showed no significant difference in S. rolfsii. In short, this study provided reliable reference genes for studying the expression and function of genes in S. rolfsii.

Antifungal activity of Ligusticum chuanxiong essential oil and its active composition butylidenephthalide against Sclerotium rolfsii.

BACKGROUND: Peanut stem rot caused by Sclerotium rolfsii is an epidemic disastrous soil-borne disease. Recently, natural products tend to be safe alternative antifungal agents to combat pathogens. RESULTS: This work determined the preliminary antifungal activity of 29 essential oils against S. rolfsii and found that Ligusticum chuanxiong essential oil (LCEO) showed the best antifungal activity, with an EC50 value of 81.79 mg L-1 . Sixteen components (98.78%) were identified in LCEO by gas chromatography-mass spectrometry analysis, the majority by volume comprising five phthalides (93.14%). Among these five phthalides, butylidenephthalide was the most effective compound against S. rolfsii. Butylidenephthalide not only exhibited favorable in vitro antifungal activity against the mycelial growth, sclerotia production and germination of S. rolfsi, but also presented efficient in vivo efficacy in the control of peanut stem rot. Seven days after application in the glasshouse, the protective and curative efficacy of butylidenephthalide at 300 mg L-1 (52.02%, 44.88%) and LCEO at 1000 mg L-1 (49.60%, 44.29%) against S. rolfsii were similar to that of the reference fungicide polyoxin at 300 mg L-1 (54.61%, 48.28%). Butylidenephthalide also significantly decreased the oxalic acid and polygalacturonase content of S. rolfsii, suggesting a decreased infection ability on plants. Results of biochemical actions indicated that butylidenephthalide did not have any effect on the cell membrane integrity and permeability but significantly decreased nutrient contents, disrupted the mitochondrial membrane, inhibited energy metabolism and induced reactive oxygen species (ROS) accumulation of S. rolfsii. CONCLUSION: Our results could provide an important reference for understanding the application potential and mechanisms of butylidenephthalide in the control of S. rolfsii. © 2023 Society of Chemical Industry.

Residue behavior and efficacy of benzothiazole in grains under different fumigation conditions.

BACKGROUND: Benzothiazole is a potential grain fumigant for Tribolium castaneum. However, its safety profile and suitable fumigation conditions remain unknown. We therefore investigated the insecticidal efficacy, accumulation and dissipation of benzothiazole in grains (wheat, corn and rice) under different temperatures. RESULTS: We established a universal detection method (modified QuEChERS coupled with GC-MS/MS) of benzothiazole residues in three grains, which provided high linearity (R2 > 0.999), sensitivity (limits of detection = 0.001 mg/kg, limits of quantification = 0.002-0.005 mg/kg), accuracy (recoveries = 88.18-118.75%) and precision (relative standard deviations < 4.78%). The insecticidal efficacy order of benzothiazole was 30 ≥ 10 > 20 °C and corn > wheat > rice. Temperature positively affected the accumulation/dissipation rate of benzothiazole. Rice was the most easily accumulated and dissipated grain for benzothiazole residues, while corn accumulated benzothiazole more than wheat but less than rice, with dissipation slower than wheat and rice. CONCLUSION: Our results provide important references for the application of benzothiazole and other fumigants. © 2023 Society of Chemical Industry.

Bioactivity of the DMI fungicide mefentrifluconazole against Sclerotium rolfsii, the causal agent of peanut southern blight.

BACKGROUND: Sclerotium rolfsii, the causal agent of peanut southern blight, has become increasingly prevalent and harmful in China, causing serious economic losses to the peanut industry. To effectively manage peanut southern blight, this study evaluated the bioactivity of the new-generation sterol demethylation inhibitor (DMI) fungicide mefentrifluconazole against peanut S. rolfsii. RESULTS: In this study, the DMI fungicide mefentrifluconazole exhibited excellent inhibitory activity against the mycelial growth of S. rolfsii, with a mean EC50 value of 0.21 ± 0.11 mg L-1 and a range of 0.02 to 0.55 mg L-1 for 261 isolates collected from Hebei, Henan and Shandong provinces. Mefentrifluconazole significantly reduced the biomass of mycelia and affected the morphology of hyphae. Although sclerotia were more tolerant to mefentrifluconazole than mycelial growth, mefentrifluconazole greatly inhibited the formation and germination of sclerotia. In addition, sclerotia produced by mefentrifluconazole-treated mycelia were deficient in nutrients (e.g., protein, carbohydrate and lipid). These results indicated that mefentrifluconazole may reduce the population of S. rolfsii in the following year. In greenhouse experiments, mefentrifluconazole showed control efficacy and good persistence against peanut S. rolfsii. The preventative and curative activities of mefentrifluconazole at 200 mg L-1 against southern blight still reached 95.36% and 60.94%, respectively, after 9 days of application. No correlation was observed for the sensitivity of S. rolfsii to mefentrifluconazole and the tested DMI, quinone outside inhibitor and succinate dehydrogenase inhibitor fungicides. CONCLUSION: All data indicated that mefentrifluconazole could provide favorable control efficacy against S. rolfsii from peanuts and reduce the infection and population of S. rolfsii in the following year. © 2023 Society of Chemical Industry.

Bioactivity and Control Efficacy of Benzovindiflupyr Against Athelia rolfsii in China.

Athelia rolfsii is a devastating soilborne pathogen that causes stem rot of peanut and severely restricts peanut production. The new generation of succinate dehydrogenase inhibitor (SDHI) fungicide benzovindiflupyr has been registered in the United States and Brazil for managing multiple plant diseases. However, it is not registered in China to control peanut stem rot. In this study, 246 isolates from major peanut production areas in Shandong, Henan, and Hebei Provinces of China were used to determine the baseline sensitivity of A. rolfsii to benzovindiflupyr. The frequency of EC50 values of benzovindiflupyr was unimodally distributed with an average EC50 of 0.12 ± 0.05 mg/liter and a range of 0.01 to 0.57 mg/liter. Benzovindiflupyr can also strongly inhibit the germination of sclerotia, with an average EC50 of 2.38 ± 1.04 mg/liter (n = 23). In addition, benzovindiflupyr exhibited great in vivo efficacy against A. rolfsii; the protective or curative efficacy (89.87%, 20.39%) of benzovindiflupyr at a concentration of 50 mg/liter was equivalent to that of the control fungicide thifluzamide at 100 mg/liter (86.39%, 16.21%). At the same concentration (e.g., 100 mg/liter), the protective efficacy (93.99%) of benzovindiflupyr was more than twice as high as the curative efficacy (45.07%). A positive correlation existed between benzovindiflupyr and isopyrazam or mefentrifluconazole, which possibly resulted from similar chemical structures or damage to the cell membrane. Our findings provide valuable information for the application of benzovindiflupyr, and the established baseline sensitivity could facilitate the monitoring and assessment of benzovindiflupyr resistance risk.

iTRAQ proteomic analysis of the inhibitory effect of 1,6-O,O-diacetylbritannilactone on the plant pathogenic oomycete Phytophthora capsici.

Phytophthora capsici is a highly destructive oomycete of vegetables; its management is challenging due to its broad host range, rapid dispersion, resilient spores and severe fungicide resistance. Identifying an effective alternative fungicide is important for the control of P. capsici. 1,6-O,O-diacetylbritannilactone (ABLOO), one of the secondary metabolites of Inula Britannica, showed a favorable inhibitory activity against P. capsici at different developmental stages, with a sensitivity order as follows: sporangia formation (30.45 mg/L) > zoospore discharge (77.69 mg/L) > mycelial growth (93.18 mg/L) > cystospore germination (591.48 mg/L). To investigate the mode of action of ABLOO in P. capsici, iTRAQ-based quantitative proteomic analysis was performed by comparing the expression levels of proteins in the control and ABLOO-treated (400 mg/L, inhibition rate of 80%) mycelial groups. A total of 65 downregulated and 75 upregulated proteins were identified in the proteomic analysis. Functional enrichment analyses showed that proteins with transmembrane transport activity were significantly inhibited, while proteins involved in energy production were significantly increased, including proteins involved in ubiquinone and other terpenoid-quinone biosynthesis, oxidative phosphorylation, and glycolysis/gluconeogenesis. The morphological results indicated that ABLOO treatment could decrease the thickness of the cell walls of P. capsici mycelia. Correspondingly, biochemical results showed that ABLOO treatment reduced the ß-1,3-glucan contents (the key component of the cell wall of P. capsici) and increased the cell membrane permeability of P. capsici. ABLOO may exhibit antioomycete activity by destroying the cell membrane of P. capsici. This study provides new evidence regarding the inhibitory mechanisms of ABLOO against P. capsici.

Baseline sensitivity and bioactivity of tetramycin against Sclerotium rolfsii isolates in Huanghuai peanut-growing region of China.

Peanut stem rot caused by Sclerotium rolfsii is a serious soil-borne disease and poses a threat to the peanut production. The antibiotic fungicide tetramycin has a broad antifungal spectrum against multiple pathogens and possess low environmental risks. In current study, a total of 250 isolates collected from Huanghuai peanut-growing region of China (Henan, Shandong and Hebei Province) were used to establish the baseline sensitivity of S. rolfsii to tetramycin. The baseline sensitivity curve was unimodal and distributed from 0.01 to 0.36 mg/L, with a mean EC50 (50% effective concentration) value of 0.11 ± 0.06 mg/L. Tetramycin also had strong inhibitory activity on the formation and germination of sclerotia. There was no significant correlation of S. rolfsii sensitivity to tetramycin and other commonly used SDHI (succinate dehydrogenase inhibitor), QoI (quinone outside respiration inhibitor) and DMI (demethylation inhibitor) fungicides. Moreover, tetramycin significantly increased the cell membrane permeability and reduced the oxalate acid content. Greenhouse experiments showed that tetramycin has both protective and curative efficacy against S. rolfsii, while protective efficacy was higher than curative efficacy. Anyhow, the bioactivity of tetramycin is similar (curative efficacy) or higher (protective efficacy) than the control fungicide validamycin. In terms of application method, root drench may be more suitable for tetramycin than spraying, because root drench of tetramycin obtained a higher efficacy. These results indicated that tetramycin may be a potential alternative fungicide for the efficient control of peanut stem rot.

Residual behavior of the potential grain fumigant 1-octen-3-ol in wheat during fumigation and ventilation processes.

BACKGROUND: Our previous works indicated that 1-octen-3-ol has promising potential as an alternative grain fumigant. However, aside from its insecticidal efficacy, the presence of 1-octen-3-ol residues in grains must be investigated to assess its food safety profile. RESULTS: A convenient and sensitive QuEChERS based GC-MS/MS method was developed to detect residues of 1-octen-3-ol in wheat. The sample pretreatment procedures were optimized. The developed method showed good linearity (R2  = 0.9999) and negligible matrix effects. The limit of detection (LOD) and limit of quantification (LOQ) for 1-octen-3-ol were 0.003 and 0.01 mg kg-1 , respectively. Recoveries at spiked concentration levels of 0.01, 0.5, 10, 100 and 200 mg kg-1 ranged from 90.8% to 112.4%, with relative standard deviations (RSDs, n = 5) ranged from 1.2 to 7.5%. In the fumigation process during wheat storage, there were positive correlations between the accumulation rate and fumigation concentration as well as between the accumulation amounts and fumigation time. In the ventilation process, temperature significantly affected the dissipation dynamics of 1-octen-3-ol in wheat, and the t1/2 values at ventilation temperatures of 30 and 5 °C for 0.1 µL mL-1 were 0.16 and 21.80 days, respectively, representing a 136-fold difference. CONCLUSION: Preservers can regulate the ventilation temperature to achieve different goals, with either a long duration period for long-term storage or rapid dissipation for quick food consumption. This study provides guidance on the reasonable usage of 1-octen-3-ol on wheat and other stored grains. © 2021 Society of Chemical Industry.

Toxicological effects of the fungal volatile compound 1-octen-3-ol against the red flour beetle, Tribolium castaneum (Herbst).

Tribolium castaneum (Herbst) is an important pest of stored grain, and benzoquinones secreted by this pest are harmful to humans. T. castaneum has developed strong resistance to fumigants, and an ecofriendly alternative for managing T. castaneum is urgently needed. 1-Octen-3-ol is a major volatile compound present in many mushrooms and fungi. In the current study, the direct toxicity and sublethal and transgenerational effects of 1-octen-3-ol on T. castaneum were investigated. Our results showed that 1-octen-3-ol had strong insecticidal activity against all developmental stages of T. castaneum and repelled T. castaneum adults. 1-Octen-3-ol showed negative effects on the development and reproduction of parental T. castaneum and the subsequent generation: LC30 and LC50 treatments significantly decreased the pupa and adult weights, pupation and emergence rates and fecundity of the parental generation. In addition, LC50 treatment shortened the larval and pupal periods. In the unexposed progeny (F1) of 1-octen-3-ol-exposed parents, decreased survival and pupation rates as well as reduced pupa and adult weights were observed under LC30 and LC50 treatments. In addition, a model food-system experiment showed that 1-octen-3-ol at 98 µL/L exhibited an efficacy of 100% after 7 days of fumigation and completely eliminated T. castaneum offspring. Although a higher concentration of 1-octen-3-ol was needed to achieve an efficacy equal to that of the positive control, dichlorvos (DDVP), 1-octen-3-ol promoted the seedling growth of wheat seeds, suggesting that the concentration used was not only acceptable but also beneficial for wheat seeds. Overall, 1-octen-3-ol seems to be a promising candidate for use as a fumigant and repellent against T. castaneum as well as a seed protectant.

Biological Activity of trans-2-Hexenal Against the Storage Insect Pest Tribolium castaneum (Coleoptera: Tenebrionidae) and Mycotoxigenic Storage Fungi.

Grain commodities in postharvest storage often deteriorate because of fungal and insect attacks. With the green consumption requirements of consumers, ecofriendly and safe pesticides are needed for grain storage. The current study investigated the efficacy of the plant volatile compound trans-2-hexenal against the storage insect pest Tribolium castaneum (Herbst) and three commonly occurring storage fungi, viz., Fusarium graminearum, Aspergillus flavus, and Aspergillus niger, to recommend its application as a botanical fumigant for grain commodities. trans-2-Hexenal weakly repels T. castaneum but has favorable insecticidal activity against multiple developmental stages of T. castaneum, ranging in sensitivity as follows: eggs (LC50 = 14.3 µl/l) > adults (31.6 µl/l) > young larvae (42.1 µl/l) > mature larvae (64.5 µl/l) > pupae (70.5 µl/l). Moreover, trans-2-hexenal caused a high malformation rate and high mortality in adults developed from fumigated pupae. In a 7-d grain, trans-2-hexenal at 0.8 µl/ml provided an appreciable efficacy (81.3%), and concentrations ≥ 0.1 µl/ml completely inhibited the offspring of T. castaneum. trans-2-Hexenal was nonphytotoxic to the seed germination and seedling growth of wheat seeds. Furthermore, trans-2-hexenal completely inhibited the growth of A. flavus, F. graminearum, and A. niger at 5, 10, and 10 µl/l, respectively. The favorable biological activity of trans-2-hexenal against T. castaneum and three frequently occurring mycotoxigenic storage fungi indicated the potential of trans-2-hexenal for simultaneously controlling pests and pathogens, which could reduce its application frequency in grains and decrease pesticide resistance risks.

Comparative Analysis of Botrytis cinerea in Response to the Microbial Secondary Metabolite Benzothiazole Using iTRAQ-Based Quantitative Proteomics.

Benzothiazole is a microbial volatile compound with strong antifungal activity against the phytopathogenic fungus Botrytis cinerea, but its mode of action against fungi remains largely unknown. Understanding the molecular mechanisms underlying its activity could aid the design and synthesis of similar compounds against pathogenic fungi. Based on the results of morphological and antifungal activity assays, B. cinerea was exposed to 2.5 µl/liter of benzothiazole for 12, 24, and 48 h, and an isobaric tags for relative and absolute quantitation-based quantitative proteomic analysis showed that 378 out of 5,110 identified proteins were differentially expressed proteins (DEPs). The majority of these DEPs were associated with carbohydrate metabolism, oxidation reduction processes, and energy production. Further analysis showed that benzothiazole inhibited mitochondrial membrane organization and decreased the mitochondrial membrane potential of B. cinerea. In addition, the key enzymes of the glyoxylate cycle were downregulated after benzothiazole treatment, and a biochemical analysis indicated that inhibition of the glyoxylate cycle by benzothiazole blocked nutrient availability and interfered with adenosine triphosphate generation. This study provides markers for future research of the molecular responses of B. cinerea to benzothiazole stress.

Development of Boscalid Resistance in Botrytis cinerea and an Efficient Strategy for Resistance Management.

Among succinate dehydrogenase inhibitors, only boscalid has been registered in China for controlling gray mold. In Shandong Province of China, it has been more than a decade since the first use of boscalid to control gray mold. In the current study, we monitored the resistance development process of Botrytis cinerea to boscalid, identified the mutation types that occurred in boscalid-resistant isolates, and proposed an original application technique to delay resistance development. A total of 720 B. cinerea isolates collected from tomato and cucumber in Shandong Province from 2014 to 2019 were determined to be sensitive to boscalid. The results showed that the sensitivity of the B. cinerea isolates to boscalid declined gradually over time, with a mean half maximal effective concentration of 0.3 ± 0.02 mg/liter in 2014 and 6.39 ± 1.66 mg/liter in 2019. The proportion of resistant isolates quickly increased from 0.81% in 2014 to 28.97% in 2019. Mutations of P225F, N230I, H272Y, and H272R in the SdhB subunit were responsible for boscalid resistance. Four concurrent mutations (G85A, I93V, M158V, and V168I) in the SdhC subunit were first discovered in Shandong Province, but they did not affect the level of boscalid resistance. Interestingly, this study found that the fruit dipping application, a precise topical application technique, could delay the development of boscalid resistance. Therefore, this application technique provides a new method for resistance management of B. cinerea.

Comparison of Transcriptome Profiles of the Fungus Botrytis cinerea and Insect Pest Bradysia odoriphaga in Response to Benzothiazole.

Benzothiazole (BT) has a strong inhibitory effect on the growth and development of a wide spectrum of fungi and insects, such as Botrytis cinerea and Bradysia odoriphaga, that cause serious losses in agriculture. To investigate the underlying antifungal and insecticidal mechanisms of BT, RNA-seq analysis was performed for B. cinerea after BT treatment for 12, 24, and 48 h and for B. odoriphaga after BT treatment for 6 and 24 h. In B. cinerea, the pectin degradation process was inhibited, suggesting a low utilization of carbohydrate sources. As the treatment time was extended, the cell walls of B. cinerea thickened, and increases in melanin synthesis and ion transport were observed. In B. odoriphaga, signaling pathways including MAPK, insulin, adipocytokine, forkhead box class O, and peroxisome proliferator-activated receptor were activated at 6 h, and phosphoenolpyruvate carboxykinase was the core gene in the signal transduction pathways that responded to BT; digestive system and melanogenesis genes were obviously altered at 24 h. In addition, we identified several insecticidal target genes, such as trypsin, aminopeptidase N, and tyrosinase. Benzothiazole significantly affected nutrient metabolism, especially carbohydrate metabolism, in both species, and the pentose and glucuronate interconversions pathway was shared by both species, although the individual genes were different in each species. Overall, our results suggested that BT was a melanogenesis disrupter for the insect but an activator for the fungus. Our findings are helpful for deeply exploring the genes targeted by BT and for developing new pesticide compounds with unique mechanisms of action.

Activity of the Novel Succinate Dehydrogenase Inhibitor Fungicide Pydiflumetofen Against SDHI-Sensitive and SDHI-Resistant Isolates of Botrytis cinerea and Efficacy Against Gray Mold.

Succinate dehydrogenase inhibitor (SDHI) fungicides are currently the most frequently used fungicides for controlling gray mold. However, isolates of Botrytis cinerea resistant to SDHI fungicides have emerged in the field. Pydiflumetofen is a new SDHI fungicide that can control a variety of fungal diseases, but its efficacy against gray mold and whether the activity of pydiflumetofen is affected by the current SDHI-resistant isolates is currently unknown. The sensitivity of 291 single-spore B. cinerea isolates collected from 2017 to 2019 to pydiflumetofen was determined by spore germination inhibition assays. The mean EC50 value (fungicide concentration resulting in a 50% inhibition compared with that of the control) of pydiflumetofen was 0.06 ± 0.01, 0.07 ± 0.02, and 0.05 ± 0.02 mg/liter in 2017, 2018, and 2019, respectively. There was no significant difference in the sensitivity of B. cinerea to pydiflumetofen among the 3 years. Furthermore, pydiflumetofen at 300 mg/liter effectively controlled gray mold on cucumber leaves (80.9%), and its efficacy was superior to that of boscalid at 400 mg/liter (42.7%). The isolates carrying P225F, N230I, H272Y, and H272R mutations in the SdhB subunit were associated with the less sensitivity of B. cinerea to SDHI fungicides. After establishing the baseline sensitivity of B. cinerea to pydiflumetofen (EC50 of 0.03 ± 0.003 mg/liter), we found that the P225F and H272Y mutant isolates showed low to moderate levels of resistance to pydiflumetofen, and the H272R and N230I mutant isolates showed low levels of resistance. The reduced sensitivity to pydiflumetofen resulted from the positive correlation of pydiflumetofen with the other four SDHI fungicides (i.e., boscalid, fluopyram, isopyrazam, and benzovindiflupyr). These results suggest that pydiflumetofen provides effective control for the management of gray mold but must be used with caution.

Evolution of the Resistance of Botrytis cinerea to Carbendazim and the Current Efficacy of Carbendazim Against Gray Mold After Long-Term Discontinuation.

Gray mold caused by Botrytis cinerea is a fungal disease that critically threatens agricultural production, and carbendazim was the first fungicide used to control B. cinerea. However, B. cinerea developed serious resistance to carbendazim, and this fungicide has thus rarely been used in the past decade in China. Due to the extended discontinuation of carbendazim use, the evolution of the resistance of B. cinerea to carbendazim in recent years is unclear, and whether carbendazim can effectively control gray mold is largely unknown. Therefore, this study determined the sensitivity of 407 B. cinerea isolates collected from 2014 to 2018 to carbendazim and the ability of carbendazim to control gray mold in the field. The results showed that the frequency of B. cinerea isolates resistant to carbendazim remained above 95%. Three different mutation types responsible for the resistance of B. cinerea to carbendazim were identified at codon 198 in the ß-tubulin gene sequence: E198V (changed from GAG to GTG), E198A (changed from GAG to GCG), and E198K (changed from GAG to AAG). Over the last 5 years, E198V was the major mutation. However, an analysis of its evolution revealed that the percentage of the E198V mutation declined after 2017 to 56.5% in 2018. In addition, the proportion of isolates with the E198K mutation decreased over time, and no isolates with this mutation were found in either 2017 or 2018. The proportion of the E198A mutation increased over the 5-year test period to reach 43.5% in 2018. Furthermore, three greenhouse experiments demonstrated that carbendazim has lost its ability to control gray mold. We attribute the above findings to our results showing that the carbendazim-resistant isolates had no fitness penalties compared with the carbendazim-sensitive isolates for sporulation and mycelial growth. In particular, the E198A mutant isolates exhibited a strong ability to sporulate, suggesting that the E198A mutation might become dominant in the future. Interestingly, the results showed that carbendazim-sensitive isolates could be easily controlled by four conventional fungicides, namely boscalid, procymidone, iprodione, and pyrimethanil, with mean EC50 values of 0.71 ± 0.2 mg liter-1, 1.33 ± 0.39 mg liter-1, 0.59 ± 0.33 mg liter-1, and 6.02 ± 3.02 mg liter-1, respectively. In conclusion, carbendazim has lost its application value and is ineffective for the control of gray mold.

Effects of benzothiazole on survival for reduced reproduction and development in Tribolium castaneum Herbst (Coleoptera: Tenebrionidae).

BACKGROUND: The red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), is an important stored-product pest that is distributed worldwide and has developed resistance to many insecticides. Identifying novel and effective alternative insecticides is important for the control of T. castaneum. The volatile compound benzothiazole has been identified as having great acute toxic activity against T. castaneum. However, a comprehensive evaluation of a new insecticide should include both direct toxic effects and sublethal effects. The aim of this study was therefore to evaluate the effects of benzothiazole on the development and reproduction of T. castaneum. RESULTS: Exposure of fourth-instar larvae to lethal and sublethal concentrations of benzothiazole (LC10 , LC30 and LC50 ) significantly decreased pupation rates, food intake and growth rates in T. castaneum. Larval duration was significantly reduced by approximately 1 day in the LC30 and LC50 treatment groups. The LC50 benzothiazole caused a significant decrease in the weight of pupae and adults, fecundity and egg hatchability. Increased and decreased nutrient (carbohydrate and lipid) contents were observed in surviving larvae and pupae, respectively. The LC30 and LC50 treatments caused the down-regulation of five growth-positive regulated genes (PI3K, AKT, CyclinE, S6K1 and S6K2) and the up-regulation of two growth-negative regulated genes (4EBP and FOXO). CONCLUSION: Benzothiazole presented adverse effects on the development and reproduction of T. castaneum, further supporting benzothiazole as a highly active compound in stored-product protection. © 2020 Society of Chemical Industry.

Evaluation of the efficacy of benzothiazole against the red flour beetle, Tribolium castaneum (Herbst).

BACKGROUND: In the context of the resistance development and health risks of currently used fumigants, it is urgent to seek more effective and ecofriendly compounds for stored-product pest control. The microbial volatile compound benzothiazole is known to have fungicidal and insecticidal activity; however, its detailed efficacy on storage pests is largely unknown. RESULTS: Benzothiazole was identified for its great ovicidal, larvicidal, pupicidal and adulticidal activity against Tribolium castaneum, and exhibited potent repellency against T. castaneum. The benzothiazole concentrations and developmental stage of T. castaneum were the key factors affecting the insecticidal effects. Adults of T. castaneum exposed to benzothiazole for as long as 168 h showed a decrease in progeny production. Based on 7 days of fumigation in the model food system, benzothiazole at 0.12 mg mL-1 provided an efficacy of 96% and completely inhibited the number of offspring. Safety profile assessment showed that benzothiazole did not affect the germination rate of wheat seeds but had a slight negative effect on seedling growth. However, sufficient ventilation and soil nutrients could relieve this adverse impact. CONCLUSION: Benzothiazole is a strong fumigant and repellent against T. castaneum. This study provides a good perspective of novel ways to control T. castaneum. © 2020 Society of Chemical Industry.

Identification and Expression Analysis of Chemosensory Receptor Genes in Bradysia odoriphaga (Diptera: Sciaridae).

The chive midge, Bradysia odoriphaga, is a major insect pest affecting Chinese chive production in China. Its adult life stage is nonfeeding and has a short life span. Hence, the perception of chemical stimuli is important for its adult behavior and reproductive success. To better understand its chemosensory process at the molecular level, chemosensory receptor genes were identified based on transcriptomes of B. odoriphaga. In total, 101 chemosensory genes were identified from the antenna and body transcriptomes, including 71 odorant receptors (ORs), 18 ionotropic receptors (IRs), 5 gustatory receptors (GRs), and 7 sensory neuron membrane proteins (SNMPs). Phylogenetic analysis indicated that most of these genes have homologs among other Dipteran insects. A transcript abundance comparison based on FPKM values was conducted to analyze the sex- and tissue-specific expression profiles of these chemosensory genes. Moreover, quantitative real-time PCR of OR transcripts was performed on different tissues (female antennae, male antennae, heads, and legs) to verify the transcriptional expression levels of ORs in the transcriptomes. This analysis suggested that 44 ORs showed significantly higher expression in the female antennae, while 16 OR transcripts were most highly expressed in the male antennae and may play significant roles in sex pheromone detection. In addition, some IRs and GRs might be involved in CO2 and sugar detection and temperature sensing. In the present study, 101 chemosensory genes were identified, and their putative functions were predicted. This work could provide a basis to facilitate functional clarification of these chemosensory genes at the molecular level.

Detection and Characterization of QoI-Resistant Phytophthora capsici Causing Pepper Phytophthora Blight in China.

Phytophthora capsici is a highly destructive plant pathogen that has spread worldwide. To date, the quinone outside inhibitor (QoI) azoxystrobin has been the choice of farmers for managing this oomycete. In this study, the sensitivity of 90 P. capsici isolates collected from Yunnan, Fujian, Jiangxi, Zhejiang, and Guangdong in southern China to azoxystrobin was assessed based on mycelial growth, sporangia formation, and zoospore discharge. Furthermore, the mitochondrial cytochrome b (cytb) gene from azoxystrobin-sensitive and -resistant P. capsici isolates was compared to investigate the mechanism of QoI resistance. The high values for effective concentration to inhibit 50% of mycelial growth and large variation factor obtained provide strong support for the existence of azoxystrobin-resistant subpopulations in wild populations. The resistance frequency of P. capsici to azoxystrobin was greater than 40%. Sensitive P. capsici isolates were strongly suppressed on V8 medium plates containing azoxystrobin supplemented with salicylhydroxamic acid at 50 µg ml-1, whereas resistant isolates grew well under these conditions. Multiple alignment analysis revealed a missense mutation in the cytb gene that alters codon 137 (GGA to AGA), causing an amino acid substitution of glycine to arginine (G137R). The fitness of the azoxystrobin-sensitive isolate is similar to that of the G137R mutant. Additionally, the P. capsici isolates used in this study exhibited decreased sensitivity to two other QoI fungicides (pyraclostrobin and famoxadone). Necessary measures should be taken to control this trend of resistance to QoI that has developed in P. capsici in southern China.