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Background Penicillium italicum (blue mold) is one of citrus pathogens causing undesirable citrus fruit decay even at strictly-controlled low temperatures (< 10 °C) during shipping and storage. P. italicum isolates with considerably high resistance to sterol demethylation inhibitor (DMI) fungicides have emerged; however, mechanism(s) underlying such DMI-resistance remains unclear. In contrast to available elucidation on anti-DMI mechanism for P. digitatum (green mold), how P. italicum DMI-resistance develops has not yet been clarified. Results The present study prepared RNA-sequencing (RNA-seq) libraries for two P. italicum strains (highly resistant (Pi-R) versus highly sensitive (Pi-S) to DMI fungicides), with and without prochloraz treatment, to identify prochloraz-responsive genes facilitating DMI-resistance. After 6 h prochloraz-treatment, comparative transcriptome profiling showed more differentially expressed genes (DEGs) in Pi-R than Pi-S. Functional enrichments identified 15 DEGs in the prochloraz-induced Pi-R transcriptome, simultaneously up-regulated in P. italicum resistance. These included ATP-binding cassette (ABC) transporter-encoding genes, major facilitator superfamily (MFS) transporter-encoding genes, ergosterol (ERG) anabolism component genes ERG2 , ERG6 and EGR11 ( CYP51A ), mitogen-activated protein kinase (MAPK) signaling-inducer genes Mkk1 and Hog1 , and Ca 2+ /calmodulin-dependent kinase (CaMK) signaling-inducer genes CaMK1 and CaMK2 . Fragments Per Kilobase per Million mapped reads (FPKM) analysis of Pi-R transcrtiptome showed that prochloraz induced mRNA increase of additional 4 unigenes, including the other two ERG11 isoforms CYP51B and CYP51C and the remaining kinase-encoding genes (i.e., Bck1 and Slt2 ) required for Slt2-MAPK signaling. The expression patterns of all the 19 prochloraz-responsive genes, obtained in our RNA-seq data sets, have been validated by quantitative real-time PCR (qRT-PCR). These lines of evidence in together draw a general portrait of anti-DMI mechanisms for P. italicum species. Intriguingly, some strategies adopted by the present Pi-R were not observed in the previously documented prochloraz-resistant P. digitatum transcrtiptomes. These included simultaneous induction of all major EGR11 isoforms ( CYP51A / B / C ), over-expression of ERG2 and ERG6 to modulate ergosterol anabolism, and concurrent mobilization of Slt2-MAPK and CaMK signaling processes to overcome fungicide-induced stresses. Conclusions The present findings provided transcriptomic evidence on P. italicum DMI-resistance mechanisms and revealed some diversity in anti-DMI strategies between P. italicum and P. digitatum species, contributing to our knowledge on P. italicum DMI-resistance mechanisms.

Background Penicillium digitatum is one of the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. The emergence of fungicide-resistant strains made the control of P. digitatum more difficult. While the genome of P. digitatum is available, there has been few reports about its resistant mechanism from the transcriptome perspective and there has been no large-scale functional annotation of the genome using expressed genes derived from transcriptomes. Methods Total RNA of  P. digitatum  strain HS-F6 (prochloraz-resistant strain) and HS-E3 (prochloraz-susceptible strain) before and after prochloraz-treatment were extracted and sequenced on an Illumina Hiseq 2000 platform. The transcriptome data of four samples were compared and analyzed using differential expression analysis, novel transcripts prediction and alternative splicing analysis, SNP analysis and quantitative real-time PCR. Results We present a large scale analysis about the transcriptome data of P. digitatum . The whole RNA was extracted from a prochloraz-resistant strain (HS-F6) and a prochloraz-susceptible strain (HS-E3) before and after prochloraz-treatment and sequenced by Illumina technology. A total of more than 100 million reads were generated and de novo assembled into 9760 transcripts that contained annotated genes after quality control and sequence assembling. 6625 single nucleotide variations (SNVs) were identified from the sequences aligned against the reference genome. Gene expression profiling analysis was performed upon prochloraz treatment in HS-F6 and HS-E3, and differential expression analysis was used to identify genes related to prochloraz-response and drug-resistance: there are 224 differentially expressed genes in HS-E3 and 1100 differentially expressed genes in HS-F6 after prochloraz-treatment. Moreover, gene expression profile in prochloraz-resistant strain HS-F6 is quite different from that in HS-E3 before prochloraz-treatment, 1520 differential expression genes were identified between the two strains. Gene ontology (GO) term enrichment and KEGG enrichment were then performed to classify the differential expression genes. Among these genes, there are a lot of transporter encoding genes including 14 MFS (Major Facilitator Superfamily) transporters, 8 ABC (ATP-binding cassette transporter) and 3 MATE (multidrug and toxic compound extrusion family) transporters. Meanwhile, the roles of typical MFS, ABC and MATE proteins in prochloraz resistance were investigated using real-time quantitative PCR. Conclusions The sequencing-based transcriptome data of P. digitatum demonstrate differences between prochloraz-resistant and prochloraz-susceptible strains with prochloraz-treatment. The differences existed in expressed transcripts, splice isoforms and GO categories, which would contribute to our knowledge on the molecular mechanisms involved in drug resistance of P. digitatum .

The effects of endocrine-disrupting chemicals (EDCs) on fertility and reproductive development represent a rising concern in modern societies. Although the neuroendocrine control of sexual maturation is a major target of EDCs, little is known about the potential role of the hypothalamus in puberty and ovulation disruption transmitted across generations. We hypothesized that developmental exposure to an environmentally relevant dose of EDC mixture could induce multi- and/or transgenerational alterations of sexual maturation and maternal care in female rats through epigenetic reprograming of the hypothalamus. We investigated the transmission of a disrupted reproductive phenotype via the maternal germline or via nongenomic mechanisms involving maternal care. Adult female Wistar rats were exposed prior to and during gestation and until the end of lactation to a mixture of the following 13 EDCs: di- -butyl phthalate (DnBP), di(2-ethylhexyl) phthalate (DEHP), bisphenol A (BPA), vinclozolin, prochloraz, procymidone, linuron, epoxynaxole, dichlorodiphenyldichloroethylene, octyl methoxynimmate, 4-methylbenzylidene camphor (4-MBC), butylparaben, and acetaminophen. Perinatally exposed offspring (F1) were mated with unexposed males to generate germ cell (F2) and transgenerationally exposed (F3 and F4) females. Sexual maturation, maternal behavior, and hypothalamic targets of exposure were studied across generations. Germ cell (F2) and transgenerationally (F3) EDC-exposed females, but not F1, displayed delayed pubertal onset and altered folliculogenesis. We reported a transgenerational alteration of key hypothalamic genes controlling puberty and ovulation ( , , and ), and we identified the hypothalamic polycomb group of epigenetic repressors as actors of this mechanism. Furthermore, we found a multigenerational reduction of maternal behavior (F1-F3) induced by a loss in hypothalamic dopaminergic signaling. Using a cross-fostering paradigm, we identified that the reduction in maternal phenotype was normalized in EDC-exposed pups raised by unexposed dams, but no reversal of the pubertal phenotype was achieved. Rats developmentally exposed to an EDC mixture exhibited multi- and transgenerational disruption of sexual maturation and maternal care via hypothalamic epigenetic reprogramming. These results raise concerns about the impact of EDC mixtures on future generations. https://doi.org/10.1289/EHP8795.

Chemical analysis shows that honey bees (Apis mellifera) and hive products contain many pesticides derived from various sources. The most abundant pesticides are acaricides applied by beekeepers to control Varroa destructor. Beekeepers also apply antimicrobial drugs to control bacterial and microsporidial diseases. Fungicides may enter the hive when applied to nearby flowering crops. Acaricides, antimicrobial drugs and fungicides are not highly toxic to bees alone, but in combination there is potential for heightened toxicity due to interactive effects. Laboratory bioassays based on mortality rates in adult worker bees demonstrated interactive effects among acaricides, as well as between acaricides and antimicrobial drugs and between acaricides and fungicides. Toxicity of the acaricide tau-fluvalinate increased in combination with other acaricides and most other compounds tested (15 of 17) while amitraz toxicity was mostly unchanged (1 of 15). The sterol biosynthesis inhibiting (SBI) fungicide prochloraz elevated the toxicity of the acaricides tau-fluvalinate, coumaphos and fenpyroximate, likely through inhibition of detoxicative cytochrome P450 monooxygenase activity. Four other SBI fungicides increased the toxicity of tau-fluvalinate in a dose-dependent manner, although possible evidence of P450 induction was observed at the lowest fungicide doses. Non-transitive interactions between some acaricides were observed. Sublethal amitraz pre-treatment increased the toxicity of the three P450-detoxified acaricides, but amitraz toxicity was not changed by sublethal treatment with the same three acaricides. A two-fold change in the toxicity of tau-fluvalinate was observed between years, suggesting a possible change in the genetic composition of the bees tested. Interactions with acaricides in honey bees are similar to drug interactions in other animals in that P450-mediated detoxication appears to play an important role. Evidence of non-transivity, year-to-year variation and induction of detoxication enzymes indicates that pesticide interactions in bees may be as complex as drug interactions in mammals.

Objectives To clone a novel major facilitator superfamily (MFS, a large protein family with diverse physiological functions in all kingdoms) transporter gene, Pdmfs2, and characterize its function in Penicillium digitatum . Results A novel MFS transporter gene, Pdmfs2 , was isolated from P. digitatum . The full-length DNA of Pdmfs2 had a 1590 bp ORF encoding a full-size MFS transporter with 529 amino acids. In a prochloraz-resistant strain (PdHS-F6), Pdmfs2 transcript level was up-regulated compared with the prochloraz-sensitive strain (PdHS-E3) and could be induced by 7 μg prochloraz/ml. The deletion of Pdmfs2 (Δ Pdmfs2 ) in PdHS-F6 led to increased susceptibility to prochloraz and lower EC 50 value (the concentration of prochloraz producing 50 % growth inhibition) compared with the PdHS-F6 or complementation strain (CO Pdmfs2 ). The Δ Pdmfs2 strain was defective in conidia yield and virulence towards citrus fruits, while the complementation of Pdmfs2 could restore the phenotypic features to a large extent. Conclusions Pdmfs2 is the second MFS transporter gene in P. digitatum and is required for prochloraz resistance, conidiation and full virulence.

Rice bakanae disease (RBD) is a typical seed-borne fungal disease caused by Fusarium fujikuroi. Prochloraz is a sterol demethylation inhibitor, which is among the most important classes of active ingredients for the management of RBD. In 2022, the total resistance frequency of F. fujikuroi to prochloraz in Zhejiang Province was 62.67%. The fitness of the prochloraz-resistant population was lower than that of the susceptible population, but its pathogenicity was slightly stronger. The S312T and F511S double mutations of Ffcyp51b were detected in the resistant isolates. Loop-mediated isothermal amplification (LAMP) technology based on S312T was established to rapidly determine prochloraz resistance in F. fujikuroi. LAMP primer mismatch design was performed based on the cyp51b gene, and 100–300 bp sequences containing a mutation at codon 312 were amplified. In a 25 µL reaction tube, 1 pg/µL DNA of F. fujikuroi could be detected. The detection limit for the frequency of prochloraz resistance was 0.498% using this method. We performed LAMP detection on rice seedlings inoculated with prochloraz-sensitive and -resistant isolates and treated them with prochloraz. Prochloraz demonstrated good control in rice seedlings. A chromogenic reaction was observed in seedlings treated with prochloraz-resistant isolates, and the results were verified using electrophoresis. It has been demonstrated that LAMP technology based on the S312T genotype can quickly and specifically detect prochloraz-resistant isolates in rice seedlings.

As an evergreen shrub, Euonymus japonicus plays a crucial role in urban landscape construction, and its growth is affected by severe foliar anthracnose caused by Colletotrichum spp. However, the biodiversity of Colletotrichum species associated with anthracnose on E. japonicus remains undetermined. This study involved a two-year collection of E. japonicus leaf samples with typical anthracnose symptoms from 9 districts in Beijing, China. A total of 194 Colletotrichum isolates were obtained, and eight Colletotrichum species were subsequently identified using morphological characteristics and molecular identification with the ACT , GADPH , CHS , TUB2 , and CAL genes, as well as the rDNA-ITS region. These species included Colletotrichum aenigma , C. fructicola , C. gloeosporioides , C. grossum , C. hebeiense , C. karstii , C. siamense , and C. theobromicola with C. siamense being the most prevalent (57%), followed by C. aenigma and C. theobromicola . Furthermore, C. fructicola , C. grossum and C. hebeiense are reported for the first time as causal agents of anthracnose on E. japonicus worldwide, and C. karstii is newly reported to be associated with E. japonicus anthracnose in China. Pathogenicity tests revealed that all tested isolates exhibited pathogenicity in the presence of wounds, emphasizing the need to avoid artificial or mechanical wounds to prevent infection in E. japonicus management. The EC 50 values of five fungicides, namely difenoconazole, flusilazole, tebuconazole, hexaconazole, and prochloraz, were found to be less than 10 mg/L, indicating their strong potential for application. Notably, the EC 50 of prochloraz was less than 0.05 mg/L for C. theobromicola . These findings offer valuable insights for the management of anthracnose on E. japonicus .

Integrating toxic fungicide into a functional stimuli-responsive nanosystem can effectively improve the fungus control specificity and reduce the effect on non-target organisms. We report here a redox and cellulase dual-responsive multifunctional nanoparticle based on bimodal mesoporous silica (BMMs) to deliver prochloraz (Pro) for the smart management of wilt disease (Pro-AC-SS-BMMs, known as P-ASB). The surface of the nanocarrier was modified with an aminosilane coupling agent, and Pro was encapsulated by physical adsorption using 2,2′-dithiodiacetic acid as a smart bridge and disulfide (SS) cross-linked aminocellulose (AC) as gatekeepers. P-ASB nanoparticles (NPs) had a spherical structure, and the size was 531.2 ± 4.9 nm. The loading rate of Pro was 28.5%, and the NPs possessed excellent redox/cellulase dual-responsive release characteristics in the presence of glutathione (GSH) and cellulase. The nanocarrier could effectively protect Pro against photodegradation and had better foliar wettability than the Pro technical. Fluorescence tracer results showed that the nanocarriers were taken up and activated by the mycelium. P-ASB NPs had better control efficacy against Rhizoctonia solani and had no significant toxicity to cells and bacteria. This study provides a new strategy for enhancing the environmental protection and promoting the development of green agriculture.

While the natural foods of the western honey bee ( Apis mellifera ) contain diverse phytochemicals, in contemporary agroecosystems honey bees also encounter pesticides as floral tissue contaminants. Whereas some ubiquitous phytochemicals in bee foods up-regulate detoxification and immunity genes, thereby benefiting nestmates, many agrochemical pesticides adversely affect bee health even at sublethal levels. How honey bees assess xenobiotic risk to nestmates as they forage is poorly understood. Accordingly, we tested nine phytochemicals ubiquitous in nectar, pollen, or propolis, as well as five synthetic xenobiotics that frequently contaminate hives—two herbicides (atrazine and glyphosate) and three fungicides (boscalid, chlorothalonil, and prochloraz). In semi-field free-flight experiments, bees were offered a choice between paired sugar water feeders amended with either a xenobiotic or solvent only (control). Among the phytochemicals, foragers consistently preferred quercetin at all five concentrations tested, as evidenced by both visitation frequency and consumption rates. This preference may reflect the long evolutionary association between honey bees and floral tissues. Of pesticides eliciting a response, bees displayed a preference at specific concentrations for glyphosate and chlorothalonil. This paradoxical preference may account for the frequency with which these pesticides occur as hive contaminants and suggests that they present a greater risk factor for honey bee health than previously suspected.

Pathogenic fungi including Penicillium digitatum and Penicillium italicum are the main destructive pathogens in the citrus industry, causing great losses during postharvest process. To our knowledge, only one mycovirus from P. digitatum has been reported, and the prevalence of such mycoviruses against citrus postharvest pathogenic fungi and their genotyping were still under investigation. In the present study, we showed that 39 of 152 Penicillium isolates from main citrus-growing areas in China were infected with various mycoviruses belonging to polymycoviruses, Narna-like viruses, and families Totiviridae , Partitivirdae and Chrysoviridae . The next generation sequencing (NGS) towards virus genome library and the following molecular analysis revealed two novel mycoviruses Penicillium digitatum polymycovirus 1 (PdPmV1) and Penicillium digitatum Narna-like virus 1 (PdNLV1), coexisting in P. digitatum strain HS-RH2. The fungicide-resistant P. digitatum strains HS-F6 and HS-E9 coinfected by PdPmV1 and PdNLV1 exhibited obvious reduction in triazole drug prochloraz resistance by mycelial growth analysis on both PDA plates and citrus fruit epidermis with given prochloraz concentration. This report at the first time characterized two novel mycoviruses from P. digitatum and revealed the mycovirus-induced reduction of fungicide resistance.