The KLF7/PFKL/ACADL axis modulates cardiac metabolic remodelling during cardiac hypertrophy in male mice.

The main hallmark of myocardial substrate metabolism in cardiac hypertrophy or heart failure is a shift from fatty acid oxidation to greater reliance on glycolysis. However, the close correlation between glycolysis and fatty acid oxidation and underlying mechanism by which causes cardiac pathological remodelling remain unclear. We confirm that KLF7 simultaneously targets the rate-limiting enzyme of glycolysis, phosphofructokinase-1, liver, and long-chain acyl-CoA dehydrogenase, a key enzyme for fatty acid oxidation. Cardiac-specific knockout and overexpression KLF7 induce adult concentric hypertrophy and infant eccentric hypertrophy by regulating glycolysis and fatty acid oxidation fluxes in male mice, respectively. Furthermore, cardiac-specific knockdown phosphofructokinase-1, liver or overexpression long-chain acyl-CoA dehydrogenase partially rescues the cardiac hypertrophy in adult male KLF7 deficient mice. Here we show that the KLF7/PFKL/ACADL axis is a critical regulatory mechanism and may provide insight into viable therapeutic concepts aimed at the modulation of cardiac metabolic balance in hypertrophied and failing heart.

Hormesis Research in China Is More Prevalent than Previously Thought.

An analysis of China's domestic publications revealed that China's hormesis-related research was enormously underestimated. China's documented hormesis-related research spans at least four decades, covers a broad spectrum of research areas, and is more abundant than previously thought. These findings should be considered in historical assessments of the concept of hormesis. Moreover, similar to the international literature, different terms have been used to describe the same phenomenon (hormesis), which hampers communication, generalization of findings and accumulation of knowledge. Hence, we advocate that 'hormesis' should be cited as a keyword in all the relevant publications written in Chinese language.

Risk and molecular mechanisms for boscalid resistance in Penicillium digitatum.

The succinate dehydrogenase inhibitor (SDHI) fungicide boscalid is an excellent broad-spectrum fungicide but has not been registered in China to control Penicillium digitatum, the causal agent of green mold of citrus. The present study evaluated the risk and molecular mechanisms for boscalid resistance in P. digitatum. Resistance induction with four arbitrarily selected sensitive isolates of P. digitatum by ultraviolet (UV) irradiation on conidia plated on boscalid-amended potato dextrose agar (PDA) and consecutive growing on boscalid-amended PDA produced five highly resistant isolates with EC50 values greater than 1000 µg/mL and two resistant isolates with EC50 lower than 200 µg/mL. Boscalid resistance of the five mutants with EC50 values above 1000 µg/mL was stable after successive transfers on PDA for 16 generations. However, for the other two mutants with EC50 lower than 200 µg/mL, the EC50 values decreased significantly after successive transfers. There was significant cross-resistance between boscalid and carboxin (r = 0.925, P < 0.001), but no significant cross-resistance was detected between boscalid and fludioxonil (r = 0.533，P = 0.095) or between boscalid and prochloraz (r = -0.543，P = 0.088). The seven resistant mutants varied greatly in the mycelia growth, sporulation, pathogenicity, and sensitivities to exogenous stresses including NaCl, salicylhydroxamic acid (SHAM), and H2O2. Alignment of the deduced amino acid sequence showed that there was no point mutation in the target enzyme succinate dehydrogenase (Sdh) subunits SdhA, SdhC, or SdhD in each of the seven resistant mutants, and the mutation of a conserved histidine residue to tyrosine (H243Y) in the subunit SdhB (i.e., iron­sulfur protein) occurred in only three highly resistant isolates. Molecular docking indicated that mutation H243Y could not prevent the binding of boscalid into the quinone-binding site of SDH in the presence of the heme moiety. However, for SDH without the heme moiety, boscalid could bind into a deeper site with a much higher affinity, and the mutation H243Y spatially blocked the docking of boscalid into the deeper site. This may be the molecular mechanism for boscalid resistance caused by SdhB-H243Y mutation.

A novel nano delivery system targeting different stages of osteoclasts.

Osteoclast (OC) abnormalities represent osteoporosis's critical mechanism (OP). OCs undergo multiple processes that range from monocytic to functional. Different drugs target OCs at different developmental stages; however, almost no Suitable drug-targeted delivery systems exist. Therefore, we designed two dual-targeting nanoparticles to target OCs at different functional stages. Using the calcitonin gene-related peptide receptor (CGRPR), which OC precursors highly express, and specific TRAPpeptides screened in the bone resorption lacuna, where mature OCs function, respectively, two types of dual-targeted nanoparticles were constructed. Afterwards, nanoparticles were grafted with hyaluronic acid (HA), which specifically binds to CD44 on the surface of the OCs. In vivo and in vitro experiments show that both nanoparticles have noticeable targeting effects on OCs. This suggests that dual-targeting nanoparticles designed for different functional periods of OC can be well targeted to the corresponding OC, and further promote the more precise delivery of drugs used to treat OP.

Sensitivity of Colletotrichum nymphaeae to Six Fungicides and Characterization of Fludioxonil-Resistant Isolates in China.

Colletotrichum nymphaeae is the dominant species causing anthracnose disease of peach in China. In this study, 140 isolates of C. nymphaeae were assessed for their sensitivity to six fungicides. It was found that C. nymphaeae was highly resistant to carbendazim, procymidone, and boscalid but sensitive to pyraclostrobin and prochloraz. For fludioxonil, the fungus exhibited differential sensitivities (i.e., approximately 14% of isolates were resistant to fludioxonil and the resistance was stable). Fludioxonil-resistant isolates had a mean EC50 value of 2.2380 µg/ml, whereas the mean EC50 value was 0.0194 µg/ml in fludioxonil-sensitive isolates. The mean EC50 values of C. nymphaeae for pyraclostrobin and prochloraz were 0.0083 µg/ml and 0.002 µg/ml, respectively. No cross-resistance was observed between fungicides from different groups. Mycelial growth rate, control efficacy, and osmotic stress responses were significantly different (P < 0.05) between fludioxonil-sensitive (FluS) and -resistant (FluR) isolates, but no significant difference was observed (P > 0.05) in virulence and sporulation between FluS and FluR isolates. No mutation was detected in coding regions of the CnOs-1, Cal, Hk1, Hog1, TPI, and Mrr1 genes. Interestingly, with fludioxonil treatment, the expression of ABC transporter gene atrB was significantly overexpressed in some resistant isolates. However, overexpression of the atrB gene was not detected in one moderately and one highly resistant isolate, indicating that other unknown mechanisms may be involved. Current findings uncovered several effective chemicals and provided the foundation for designing management strategies to practically control peach anthracnose with the most effective demethylation inhibitor fungicides and quinone outside inhibitor fungicides.

Sensitivity of Colletotrichum fructicola and Colletotrichum siamense of Peach in China to Multiple Classes of Fungicides and Characterization of Pyraclostrobin-Resistant Isolates.

Anthracnose, mainly caused by Colletotrichum gloeosporioides species complex including Colletotrichum fructicola and Colletotrichum siamense, is a devastating disease of peach. Chemical control has been widely used for years, but management failures have increased with the commonly used fungicides. Therefore, screening of sensitivity of Colletotrichum spp. to fungicides with different modes of action is needed to make proper management strategies for peach anthracnose. In this study, the sensitivity of 80 isolates of C. fructicola and C. siamense was screened for pyraclostrobin, procymidone, prochloraz, and fludioxonil based on mycelial growth inhibition at discriminatory doses. Results showed that C. fructicola and C. siamense isolates were highly resistant to procymidone and fludioxonil with 100% resistance frequencies to both fungicides, but sensitive to prochloraz, i.e., no resistant isolates were found. For pyraclostrobin, 74% of C. fructicola isolates showed high resistance, 26% showed low resistance, and all of the C. siamense isolates showed low resistance. No positive cross-resistance was observed between pyraclostrobin and azoxystrobin even when they are members of the same quinone outside inhibitor (QoI) fungicide group or between pyraclostrobin and non-QoIs. Resistant isolates to QoI fungicides were evaluated for the fitness penalty. Results showed that no significant differences except for the mycelial growth rates that were detected between high- and low-resistance isolates of C. fructicola. Molecular characterization of the Cyt b gene revealed that the G143A point mutation was the determinant of the high resistance in C. fructicola. This study demonstrated the resistance status of C. fructicola and C. siamense to different fungicides and briefly discussed implications of that resistance. Demethylation inhibitor fungicides were found to be the best option among the different chemicals studied here, to control peach anthracnose in China.

Dual stimuli-responsive fungicide carrier based on hollow mesoporous silica/hydroxypropyl cellulose hybrid nanoparticles.

The controlled release of pesticides based on nanoparticle platforms has emerged as a new technology for increasing the efficiency of pesticides and for reducing environmental pollution because of their size-dependent and target-modifying properties. In the present study, pH/cellulase dual stimuli-responsive controlled-release formulations (PYR-HMS-HPC) were designed by grafting hydroxypropyl cellulose onto pyraclostrobin-loaded hollow mesoporous silica nanoparticles via an ester linkage. The PYR-HMS-HPC formulations were characterized by Fourier transform infrared spectroscopy, thermogravimetric analyzer, transmission electron microscope and scanning electron microscope. The results demonstrated that PYR-HMS-HPC with a loading capacity of 12.1 wt% showed excellent pyraclostrobin release behaviors in response to acidic environments and the introduction of cellulase, could effectively prevented pyraclostrobin from photolysis. Compared with commercial pyraclostrobin formulations, the PYR-HMS-HPC formulations showed much stronger and statistically significant fungicidal activity against Magnaporthe oryzae from 7 to 21 days. Furthermore, the Allium cepa chromosome aberration assay demonstrated that the PYR-HMS-HPC formulations reduced the genotoxicity of pyraclostrobin. These pH/cellulase dual stimuli-responsive controlled-release formulations are of great interest for sustainable on-demand crop disease protection.

Phylogenetic and Haplotype Network Analyses of Diaporthe eres Species in China Based on Sequences of Multiple Loci.

Diaporthe eres is considered one of the most important causal agents of many plant diseases, with a broad host range worldwide. In this study, multiple sequences of ribosomal internal transcribed spacer region (ITS), translation elongation factor 1-α gene (EF1-α), beta-tubulin gene (TUB2), calmodulin gene (CAL), and histone-3 gene (HIS) were used for multi-locus phylogenetic analysis. For phylogenetic analysis, maximum likelihood (ML), maximum parsimony (MP), and Bayesian inferred (BI) approaches were performed to investigate relationships of D. eres with closely related species. The results strongly support that the D. eres species falls into a monophyletic lineage, with the characteristics of a species complex. Phylogenetic informativeness (PI) analysis showed that clear boundaries could be proposed by using EF1-α, whereas ITS showed an ineffective reconstruction and, thus, was unsuitable for speciating boundaries for Diaporthe species. A combined dataset of EF1-α, CAL, TUB2, and HIS showed strong resolution for Diaporthe species, providing insights for the D. eres complex. Accordingly, besides D. biguttusis, D. camptothecicola, D. castaneae-mollissimae, D. cotoneastri, D. ellipicola, D. longicicola, D. mahothocarpus, D. momicola, D. nobilis, and Phomopsis fukushii, which have already been previously considered the synonymous species of D. eres, another three species, D. henanensis, D. lonicerae and D. rosicola, were further revealed to be synonyms of D. eres in this study. In order to demonstrate the genetic diversity of D. eres species in China, 138 D. eres isolates were randomly selected from previous studies in 16 provinces. These isolates were obtained from different major plant species from 2006 to 2020. The genetic distance was estimated with phylogenetic analysis and haplotype networks, and it was revealed that two major haplotypes existed in the Chinese populations of D. eres. The haplotype networks were widely dispersed and not uniquely correlated to specific populations. Overall, our analyses evaluated the phylogenetic identification for D. eres species and demonstrated the population diversity of D. eres in China.

Regulation of fast and slow light characteristics of the add-drop ring-resonator employing an assisted ring.

We demonstrate theoretically and experimentally that the fast and slow light characteristics of the add-drop ring-resonator (ADRR) can be regulated by introducing an assisted ring. This novel geometry is named ring-assisted add-drop ring-resonator (RA-ADRR). When the assisted ring is under-coupled, the fast and slow light characteristics of through and drop ports of the RA-ADRR will be reversed, which is different from the coupled resonator induced transparency (CRIT) studied previously. With the decrease of loss, the dispersion peak (dip) of the two ports will grow up towards the opposite directions and finally the inversion occurs. Meanwhile, we find that by increasing the circumference of the assisted ring, the dispersion of the two ports could be improved proportionally. The experimental results show that the maximum group delays of the through and drop ports are 115 ns and -485 ns, respectively. This novel phenomenon could greatly enhance the sensitivity of slow light interferometers and also has potential applications in optical communication, network, filtering and switching.

Baseline sensitivity and fungicidal action of propiconazole against Penicillium digitatum.

Green mold, caused by Penicillium digitatum, is the most important citrus postharvest disease worldwide and often causes substantial economic losses to the citrus industry. The demethylation inhibitor (DMI) fungicides are highly effective against a broad range of fungal pathogens, but the DMI fungicide propiconazole has not been registered yet in China for the control of citrus green mold. In this study, baseline sensitivity of P. digitatum to propiconazole was determined. The frequency distribution of logarithms of EC50 values for 118 isolates collected from five regions in China was bimodal, and among the 118 isolates, 18 isolates were less sensitive or had low resistance to propiconazole. The mean EC50 value of the sensitive 100 isolates was 0.104 mg/L. Preventive control efficacies on Satsuma mandarin for propiconazole at 200 and 400 mg/L were 63.1 and 84.3%, respectively. The fruit treated with propiconazole at 40 and 100 mg/L produced significantly fewer conidia, and the virulence of the conidia decreased by 12.3 and 14.8%, respectively. Studies with propidium iodide showed that the membrane integrity was damaged for 25.6% of conidia produced on PDA amended with propiconazole at 0.1 mg/L. Fluorescence microscopy observations of P. digitatum conidia stained with 2,7-dichlorofluorescin showed that propiconazole significantly induced the generation of intracellular reactive oxygen species (ROS). Compared with the sensitive isolates, no point mutations were detected in either the coding or promoter region of the target gene CYP51A of the isolates with low resistance to propiconazole. However, the relative expression levels of CYP51A for three resistant isolates were higher than sensitive isolates, and the mean relative expression was 2.08 for resistant isolates versus 0.62 for sensitive isolates in the absence of propiconazole and 3.12 versus 1.44 in the presence of propiconazole. These results indicate increased expression of CYP51A is the molecular mechanism for low resistance of P. digitatum to propiconazole.

Pyrimethanil Sensitivity and Resistance Mechanisms in Penicillium digitatum.

Pyrimethanil is an anilinopyrimidine (AP) fungicide that is highly effective in controlling green mold caused by Penicillium digitatum but has not yet been registered in China to control postharvest diseases of citrus. In this study, baseline sensitivity of P. digitatum to pyrimethanil was established based on the effective concentrations for 50% inhibition (EC50) values of 127 isolates collected from five major citrus-growing regions of China. The distribution of these EC50 values was unimodal but with a long right tail. The mean ± SD EC50 value was 0.137 ± 0.046 µg/ml, and the minimum and maximum were 0.073 and 0.436 µg/ml, respectively. Pyrimethanil in potato dextrose agar (PDA) at 0.20 µg/ml decreased methionine production in the mycelia by 21.6% and reduced the activity of cell wall-degrading enzymes cellulase and pectinase by 9.1 and 32.8%, respectively. Twelve pyrimethanil-resistant mutants were obtained by consecutive subculturing of 12 arbitrarily selected sensitive isolates on pyrimethanil-amended PDA for four generations, and the resistance factors ranged from 69 to 3,421. There was no cross-resistance between pyrimethanil and prochloraz (r = 0.377, P = 0.123). Compared with their parental isolates, pyrimethanil-resistant mutants had reduced pathogenicity to citrus fruit but higher tolerance to hydrogen peroxide. No differences were detected in tolerance to NaCl, CaCl2, Congo red, or sodium dodecyl sulfate. The exogenous addition of methionine into PDA partially alleviated pyrimethanil toxicity to the sensitive isolates but had no significant effect on toxicity to the resistant mutants. Sequencing of cystathionine γ-synthase encoding genes CGS1 and CGS2, the potential target genes for pyrimethanil, showed that there was no nucleotide mutation in the coding region of CGS of the pyrimethanil-resistant mutants. However, the relative expression of CGS1 and CGS2 of the pyrimethanil-resistant mutants was reduced by 42.5 and 57.4%, respectively. These results have important implications for applications of pyrimethanil to control P. digitatum and for understanding the modes of action and resistance mechanisms of pyrimethanil.

Hormetic Effects of Dimethachlone on Mycelial Growth and Virulence of Sclerotinia sclerotiorum.

Fungicide hormesis has implications for the application of fungicides to control plant diseases. We investigated the hormetic effects of the dicarboximide fungicide dimethachlone on mycelial growth and virulence of the necrotrophic plant pathogen Sclerotinia sclerotiorum. Dimethachlone at sublethal doses in potato dextrose agar (PDA) increased the mycelial growth of S. sclerotiorum. After the growth-stimulated mycelia were subcultured on fresh PDA and inoculated on rapeseed leaves, increased mycelial growth and virulence were observed, indicating that hormetic traits were passed down to the next generation. Dimethachlone applied to leaves at 0.002 to 500 µg/ml stimulated virulence, with a maximum stimulation amplitude (MSA) of 31.4% for the isolate HLJ4, which occurred at 2 µg/ml. Dimethachlone-resistant isolates and transformants had a mean virulence MSA of 30.4%, which was significantly higher (P = 0.008) than the MSA for sensitive isolates (16.2%). Negative correlations were detected between MSA and virulence in the absence of any fungicide (r = -0.872, P < 0.001) and between MSA and mycelial growth on PDA (r = -0.794, P = 0.002). Studies on hormetic mechanisms indicated that dimethachlone had no significant effects on expression levels of three virulence-associated genes, that is, a cutinase-encoding gene SsCut, a polygalacturonase gene SsPG1, or an oxaloacetate acetylhydrolase gene SsOah1. The results will contribute to understanding hormesis and have implications for the judicious application of fungicides to control plant diseases.

Fungicidal Actions and Resistance Mechanisms of Prochloraz to Penicillium digitatum.

The demethylation inhibitor (DMI) fungicide prochloraz has been widely used in China to control citrus green mold, which is caused by Penicillium digitatum. The 50% effective concentration (EC50) values of prochloraz for 129 isolates of P. digitatum collected in 2017 from citrus groves of four provinces of China ranged from 0.0032 to 0.4582 mg/liter. Analysis of the distribution of natural logarithms of EC50 values indicated that 111 isolates with EC50 values lower than 0.05 mg/liter could be considered sensitive to prochloraz. Relative baseline sensitivity was established based on the 111 sensitive isolates, and the mean EC50 value was 0.0090 ± 0.0054 mg/liter (SD). Prochloraz at 60, 100, and 140 mg/liter provided preventive efficacies of 67.8, 93.0, and 96.4%, respectively. Prochloraz at 0.005 and 0.01 mg/liter disrupted cell membrane integrity of conidia but reduced cell membrane permeability of mycelia. Prochloraz at 0.01 mg/liter reduced ergosterol content in mycelia by 41.8%. Two prochloraz-resistant isolates with EC50 values of 3.97 and 5.68 mg/liter were attained by consecutive subculturing on prochloraz-amended PDA. Studies on the expression levels of three potential target genes, CYP51A, CYP51B, and CYP51C, demonstrated that whether in the absence or presence of prochloraz, only CYP51B in the resistant isolates was overexpressed at least 10-fold higher than that of the sensitive ones. Sequencing of the three genes showed that only CYP51B in the resistant isolates had a 199-bp insertion in the promoter region. In addition, only CYP51B displayed point mutations of G405S, G389C, and Y390S in the coding regions in the resistant isolates. These results were important for understanding the resistance mechanisms of P. digitatum to prochloraz.

METTL3 is essential for postnatal development of brown adipose tissue and energy expenditure in mice.

Brown adipose tissue (BAT) undergoes rapid postnatal development and then protects against cold and obesity into adulthood. However, the molecular mechanism that determines postnatal development and maturation of BAT is largely unknown. Here we show that METTL3 (a key RNA methyltransferase) expression increases significantly in interscapular brown adipose tissue (iBAT) after birth and plays an essential role in the postnatal development and maturation of iBAT. BAT-specific deletion of Mettl3 severely impairs maturation of BAT in vivo by decreasing m6A modification and expression of Prdm16, Pparg, and Ucp1 transcripts, which leads to a marked reduction in BAT-mediated adaptive thermogenesis and promotes high-fat diet (HFD)-induced obesity and systemic insulin resistance. These data demonstrate that METTL3 is an essential regulator that controls iBAT postnatal development and energy homeostasis.

Stimulatory Effects of Boscalid on Virulence of Sclerotinia sclerotiorum Indicate Hormesis May Be Masked by Inhibitions.

Hormetic effects of fungicides on phytopathogens are of great importance for proper application of fungicides. The aim of the present study was to investigate the stimulatory effects of the fungicide boscalid on mycelial growth and virulence of the devastating plant pathogen Sclerotinia sclerotiorum. Boscalid in potato dextrose agar (PDA) at a dosage range from 0.0005 to 0.002 µg/ml exerted statistically significant (P ≤ 0.015) stimulations on mycelial growth of S. sclerotiorum, and the maximum stimulation magnitudes were 5.55 ± 0.73% (mean ± SD) for the four isolates tested. Boscalid in PDA at 0.02 µg/ml inhibited mycelial growth of isolates HLJ3H and HLJ4H by 15.0 and 8.9%, respectively. However, after the growth-inhibited mycelia were inoculated on rapeseed leaves, isolates HLJ3H and HLJ4H exhibited virulence stimulations of 8.7 and 17.8%, respectively, indicating that hormesis may be masked by inhibitions. Boscalid sprayed at 0.0001 to 0.1 µg/ml on detached rapeseed leaves had significant (P ≤ 0.041) stimulations on virulence of S. sclerotiorum, and the maximum stimulation magnitudes were 17.90 ± 5.94% (mean ± SD) for the four isolates tested. Experiments on 12 isolates with different levels of virulence showed there was a negative correlation (R = -0.663, P = 0.019) between the maximum virulence stimulation magnitude and virulence of S. sclerotiorum in the absence of fungicide. Boscalid at stimulatory concentrations had no significant effect on the expression levels of three virulence-associated genes that encode cutinase (SsCut), polygalacturonase (SsPG1), and oxaloacetate acetylhydrolase (SsOah1). The molecular mechanisms for hormetic effects of boscalid on S. sclerotiorum remain to be studied in the future.

Magnetic-field tuning whispering gallery mode based on hollow microbubble resonator with Terfenol-D-fixed.

We propose a method of magnetic-field tuning whispering gallery modes (WGMs) based on a hollow microbubble resonator (HMBR) with Terfenol-D-fixed. WGMs are excited by the evanescent field from a tapered fiber coupling with an HMBR. Both ends of the HMBR are fixed with Terfenol-D and vary with different lengths of the Terfenol-D. The length of the Terfenol-D varies with the external magnetic field for the high magnetostriction coefficient of Terfenol-D. The magnetic field sensitivity of 0.081 pm/mT in the magnetic field range of 0.14 mT-21.8 mT is achieved. The $Q$Q-factor of the HMBR can be regulated up to ${2.07} \times {{10}^4}$2.07×104 with physical stretching HMBR. This work provides a novel tuning whispering gallery mode scheme and a broad application prospect in the fields of optical measurement and precise optical clocks in the future.

Stimulatory Effects of Sublethal Doses of Carbendazim on the Virulence and Sclerotial Production of Botrytis cinerea.

Stimulatory effects of low doses of fungicides on the virulence of phytopathogens have profound implications for applications of fungicides. The present study demonstrated that carbendazim sprayed at 0.001 to 0.03 µg/ml had stimulatory effects on the virulence of mycelia of Botrytis cinerea, and the maximum percent stimulations were 15.5 and 21.4% for isolates HB459 and HB536, respectively. Potato dextrose agar (PDA) amended with carbendazim at 0.01, 0.02, and 0.05 µg/ml inhibited mycelial growth of isolate HB536 by 0.8, 10.0, and 30.6%, respectively. However, after the inhibited mycelia were inoculated on cucumber leaves, virulence increased by 10.1, 12.9, and 10.8%, respectively. With respect to sclerotial production, carbendazim at 0.005 and 0.02 µg/ml in PDA significantly (P < 0.05) increased, while at 0.1 µg/ml significantly (P < 0.05) reduced the sclerotial number and weight of both isolates compared with nontreated controls. Conidia germination percentages slightly yet statistically significantly (P < 0.05) increased after being inoculated on PDA amended with carbendazim at 0.001 and 0.005 µg/ml. Carbendazim at 0.001∼0.02 µg/ml, either sprayed on cucumber leaves or cosuspended with conidia, exerted significantly (P < 0.05) stimulatory effects on the virulence of B. cinerea conidia. Mechanism studies showed that sublethal doses of carbendazim did not increase the expression levels of pathogenicity-related pectin methylesterase gene Bcpme1, endopolygalacturonase gene Bcpg2, cutinase gene CutA, xylanase gene Xyn11A, or NADPH oxidase gene BcnoxA.

Effects of SHAM on the Sensitivity of Sclerotinia sclerotiorum and Botrytis cinerea to QoI Fungicides.

It is a common practice to add salicylhydroxamic acid (SHAM) into artificial medium in the in vitro sensitivity assay of fungal phytopathogens to the quinone outside inhibitor (QoI) fungicides. The rationale for adding SHAM is to inhibit fungal alternative oxidase, which is presumed to be inhibited by secondary metabolites of plants. Therefore, the ideal characteristics of SHAM should be almost nontoxic to phytopathogens and have no significant effect on control efficacy of fungicides. However, this study showed that the average effective concentration for 50% inhibition (EC50) of mycelial growth values of SHAM were 97.5 and 401.4 µg/ml for Sclerotinia sclerotiorum and Botrytis cinerea, respectively. EC50 values of the three QoI fungicides azoxystrobin, kresoxim-methyl, and trifloxystrobin in the presence of SHAM at 20 and 80 µg/ml for S. sclerotiorum and B. cinerea, respectively, declined by 52.7 to 78.1% compared with those without SHAM. For the dicarboximide fungicide dimethachlone, the average EC50 values in the presence of SHAM declined by 18.2% (P = 0.008) for S. sclerotiorum and 35.9% (P = 0.012) for B. cinerea. Pot experiments showed that SHAM increased control efficacy of the three QoI fungicides against the two pathogens by 43 to 83%. For dimethachlone, SHAM increased control efficacy by 134% for S. sclerotiorum and 86% for B. cinerea. Biochemical studies showed that SHAM significantly inhibited peroxidase activity (P = 0.024) of B. cinerea and esterase activity (P = 0.015) of S. sclerotiorum. The strong inhibitions of SHAM per se on mycelial growth of B. cinerea and S. sclerotiorum and significant influences on the sensitivity of the two pathogens to both the QoI fungicides and dimethachlone as well as inhibitions on peroxidase and esterase indicate that SHAM should not be added in the in vitro assay of sensitivity to the QoI fungicides.

Hormetic Effects of Mixtures of Dimethachlone and Prochloraz on Sclerotinia sclerotiorum.

Previous studies showed that dimethachlone has significant hormetic effects on phytopathogenic fungus Sclerotinia sclerotiorum. The present study investigated hormetic effects of mixtures of dimethachlone and prochloraz on mycelial growth and virulence of two dimethachlone-resistant isolates of S. sclerotiorum. The stimulatory dimethachlone dosage range was around 1 to 100 µg/ml in potato dextrose agar (PDA) medium for mycelial growth of the two isolates, and dimethachlone at 10 and 50 µg/ml had the maximum percent stimulations of 80.6 and 19.3% for isolates JMS14 and HLJ4, respectively. Prochloraz at 0.0003 and 0.002 µg/ml had the maximum percent stimulations of 9.3 and 11.1% for isolates JMS14 and HLJ4, respectively. However, dimethachlone and prochloraz mixed at their respective stimulatory concentrations had the maximum percent stimulations of 48.1 and 9.3% for isolates JMS14 and HLJ4, respectively. After the mycelia with increased and inhibited growth on fungicide-amended PDA were subcultured on PDA without fungicide, mycelial growth for the second generation increased compared with the nontreated control. After the mycelia grown on fungicide-amended PDA were inoculated on rapeseed leaves, the amplitude of virulence stimulation was much greater than that of mycelial growth on PDA amended with fungicide, and the inhibited mycelia also showed substantially increased virulence on leaves. The mixture of dimethachlone at 100 µg/ml and prochloraz at 0.03 µg/ml in PDA inhibited mycelial growth of isolate JMS14 by 59.4%; however, after the inhibited mycelia were inoculated on rapeseed leaves, virulence was stimulated by 69.0%. Spraying sublethal doses of dimethachlone and prochloraz on rapeseed leaves also exhibited significant stimulatory effects on virulence. For isolate JMS14, the stimulatory concentration ranges for dimethachlone and prochloraz were around 1 to 600 µg/ml and 0.0003 to 0.18 µg/ml, respectively. The fitted curve of virulence stimulation for the mixture of dimethachlone and prochloraz shifted to the left on the x axis, denoting dose-additive interactions between the two fungicides with regard to virulence stimulation. Spraying dimethachlone alone at 10 to 50 µg/ml had significant stimulations on virulence, whereas prochloraz alone at 10 to 50 µg/ml had significant inhibitory effects on virulence, and the mixture of dimethachlone and prochloraz at the concentration ratio of 1:1 had greater inhibitory effects than prochloraz alone, indicating dose-additive interactions for the inhibitory effects. Dimethachlone and prochloraz and their mixtures increased tolerance of mycelia to hydrogen peroxide. Dimethachlone significantly increased, whereas prochloraz reduced mycelial cell membrane permeability, and the mixture of the two fungicides had effect-additive interactions with respect to effects on cell membrane permeability. These studies will advance our understanding of hormesis of fungicide mixtures.

Hormetic Effects of Mixtures of Carbendazim and Iprodione on the Virulence of Botrytis cinerea.

Hormetic effects of fungicides on mycelial growth and virulence of plant pathogenic fungi have been reported, but the effects of fungicide mixtures on virulence hormesis of plant pathogens remain to be investigated. In this study, hormetic effects of mixtures of carbendazim and iprodione on the virulence of two carbendazim-resistant isolates of Botrytis cinerea were determined. Spraying carbendazim alone at 3 to 800 µg/ml exhibited hormetic effects on virulence to cucumber leaves, and carbendazim at 10 µg/ml had the maximum stimulation of 16.7% for isolate HBtom451. Spraying iprodione alone at 0.0001 to 0.0625 µg/ml exhibited hormetic effects on virulence, and iprodione at 0.025 µg/ml had the maximum stimulation of 18.7% for isolate HBtom451. However, spraying simultaneously carbendazim at 800 µg/ml and iprodione at 0.0625 µg/ml showed inhibitory effects on virulence to cucumber leaves. The mixture of carbendazim at 3 µg/ml and iprodione at 0.0001 µg/ml had much higher virulence stimulations than either fungicide at the same concentration alone. The maximum stimulation for the mixtures occurred at 10 and 0.0005 µg/ml for carbendazim and iprodione, respectively, and these concentrations were much lower than the concentration of their respective fungicide alone eliciting the maximum stimulations. The maximum stimulation amplitude for the mixture was slightly higher than that of each fungicide alone. These results demonstrated that carbendazim and iprodione mainly had dose-additive rather than amplitude-additive interactions when sprayed simultaneously with regard to virulence stimulations. Studies on virulence stimulations for mycelia treated with fungicide in potato dextrose agar showed that the maximum stimulation for the mixtures occurred at concentrations much lower than the concentration of carbendazim alone, indicating a dose-additive interaction when compared with carbendazim hormesis. Studies on potential physiological mechanisms of hormesis showed that increased tolerance to H2O2 may be one of the mechanisms for virulence hormesis for the mixtures of iprodione and carbendazim. These studies will advance our understanding of hormesis of fungicide mixtures.