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Laccases are multicopper oxidases (E.C. 1.10.3.2) that catalyze the oxidation of many phenolic compounds. In this study, a novel laccase, Stlac4, from Setosphaeria turcica was cloned and expressed in Escherichia coli by insertion into the pET-30a expression plasmid. The recombinant laccase was purified and visualized on SDS-PAGE as a single band with an apparent molecular weight of 71.5 KDa, and confirmed by Western blot. The maximum activity of the purified laccase was 127.78 U · mg−1, the optimum temperature and pH value were 60 °C and 4.0 respectively, measured by oxidation of 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS). Purified laccase activity under different metal ions and an inhibitor were tested, revealing that laccase activity increased by approximately 434.8% with Fe3+, and 217.4% with Cu2+ at 10 mmol · L−1 concentrations, Mn2+ increased the laccase activity only at 5 mmol · L−1, while Na+ increased activity at 1 mmol · L−1 but inhibited activity at 5 and 10 mmol · L−1. SDS increased laccase activity at 1 mmol · L−1, and inhibited activity at 5 and 10 mmol · L−1. [ABSTRACT FROM AUTHOR]

A total of 225 maize kernel samples were collected from Shandong Province in China from 2012 to 2014 and analysed for contamination withFusariumspp. and fumonisins (FBs) using molecular methods and high-performance liquid chromatography with fluorescence detection. The results showed that the average incidences ofFusariumspp. in 2012, 2013 and 2014 were 23.3%, 37.1% and 36.5%, respectively,Fusarium verticillioidesbeing the predominant species. In 2012, the average contamination level of FBs was 3071 ng g−1, which was higher than that in 2014 (2913 ng g−1) and 2013 (2072 ng g−1). Of all samples, 13% and 19% had FB contamination levels higher than 2000 and 4000 ng g−1, which are the maximum limits as set by the Food and Drug Administration of the United States and the European Commission, respectively. Therefore, efforts should be taken to minimise the potential risk of FBs to the health of humans and animals. [ABSTRACT FROM AUTHOR]

Fusariumspecies, which can produce mycotoxins, are the predominant pathogens causing maize ear rot, a disease that results in severe economic losses and serves as a potential health risk for humans and animals. A survey was conducted in 2012 to investigate the contamination of maize byFusariumspecies and fumonisins B1and B2. A total of 250 maize samples were randomly collected from nine provinces (Hebei, Shanxi, Inner Mongolia, Yunnan, Sichuan, Guizhou, Heilongjiang, Liaoning and Ningxia) in China.Fusariumspecies were isolated and identified using morphological (electron microscope) and molecular methods (polymerase chain reaction (PCR) and sequencing). Fumonisins B1and B2were analysed using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) with OPA (2-Mercaptoethanol, o-phthaldialdehyde) post-column derivatisation. A total of 2321Fusariumisolates (20.7%) were obtained from all the samples. These isolates included nineFusariumspecies, namely,F. graminearum,F. verticillioides,F. subglutinans,F. proliferatum,F. temperatum,F. oxysporum,F. equiseti,F. meridionaleandF. chlamydosporum. The incidence of occurrence ofFusariumspecies in Guizhou was the highest, while in Inner Mongolia it was the lowest.F. verticillioideswas the dominant species of maize ear rot in Liaoning, Sichuan, Hebei and Ningxia.F. graminearumwas the dominant species in Yunnan, Guizhou and Shanxi.F. subglutinanswas the dominant species in Heilongjiang.F. verticillioidesandF. graminearumpercentages were the same in Inner Mongolia. The incidence of fumonisins in Liaoning was high (up to 81.0%) and in Heilongjiang low (up to 10.3%). Except Shanxi, more than 50% of maize samples from other provinces were contaminated with fumonisins, with concentrations less than 500 ng g−1. About 33% of maize samples from Yunnan were contaminated with high levels of fumonisins, and average of fumonisin levels were 5191 ng g−1.Fusariumspecies causing maize ear rot in different areas in China were highly diverse and such areas with exposure to high levels of fumonisin contamination have a potential health risk for human and animals. [ABSTRACT FROM PUBLISHER]

A total of 125 maize kernel samples were collected from Hebei province in China during 2011–2013 and were analysed for incidence and contamination levels of fumonisins (FB1 + FB2) by HPLC with fluorescence detection. The incidence of FBs for all samples was 46.4%. The mean contamination level of FBs for the samples collected in 2013 was 706 μg kg−1, which was higher than the levels in 2012 (429 μg kg−1) and 2011 (250 μg kg−1). All samples, except five, exhibited total FB levels below 4000 μg kg−1, which is the maximum limit as set by the European Commission. The probable daily intakes of FBs (0.04 in 2011; 0.07 in 2012; 0.12 in 2013, expressed as μg kg−1body weight/day) were all within the provisional maximum tolerable daily intake of 2.0 μg kg−1of body weight/day as set by the Joint FAO/WHO Expert Committee on Food Additives. Nevertheless, monitoring is needed to prevent and control the potential risk of FB exposure to the consumers. [ABSTRACT FROM PUBLISHER]

Fumonisin B1(FB1) and fumonisin B2(FB2) are the most abundant fumonisins (FBs) occurring worldwide in maize, infected mainly byFusarium verticillioidesandF. proliferatum. A total of 307 corn kernel samples were collected from 45 districts of Gansu, Shandong, Ningxia and the Inner Mongolia provinces of the north and northwest China. The samples were analysed for FB1and FB2by high-performance liquid chromatography. The FBs (FB1+ FB2) incidence rate in samples from Gansu, Shandong, Ningxia and Inner Mongolia were 31.5%, 81.1%, 46.2% and 53.6%, respectively. Average FBs concentration was 703 μg/kg and the concentrations ranged from ≤11 to 13,110 μg kg−1. Results were compared with the European Commission (EC) regulation for FB1+ FB2in unprocessed maize for human consumption of 4 mg kg−1. Contamination in 17 samples was higher than these levels. More than 80% of the samples from Liaocheng county, which is located in the Shandong province, were contaminated with FBs, with a mean total FB concentration of 2496 μg/kg. The result was significantly different from that of the Inner Mongolia (1399 μg/kg), Ningxia (373 μg/kg) and Gansu (175 μg/kg). Average exposure to FBs (0.12 μg/kg body weight/day) is within the provisional maximum tolerable daily intake of 2.0 mg/kg of body weight set by the Joint Food and Agriculture Organization and World Health Organization Expert Committee on Food Additives. [ABSTRACT FROM AUTHOR]

Cyclic adenosine monophosphate (c AMP)-dependent protein kinase A ( PKA) is an important mediator of signal transduction in eukaryotic cells. Thus, identifying its function is necessary to understand the c AMP signaling network. St PKA-c, the PKA catalytic subunit gene in Setosphaeria turcica, was investigated by RNA interference technology. Transformant strains M3, M5, and M9 with diverse St PKA-c silencing efficiency were confirmed by reverse transcription polymerase chain reaction and Northern blot. Compared with the wild-type strain 01-23, the transformant strains exhibited increased growth rate and significantly decreased conidium production. In addition, the ratios of spore germination and appressorium formation and penetration were slightly reduced. Relative to the wild-type strain, the transformants demonstrated different colony color, greatly reduced pathogenicity, and similar HT-toxin activity. Further studies showed that the content of intracellular melanin in the transformants significantly decreased, and the transcription of transcriptional factor St MR was down-regulated correspondingly. The transcription and enzyme activity of xylanase was also impaired. Thus, we proposed that St PKA-c was mainly involved in the mycelium growth, conidiation, and pathogenesis of S. turcica. Furthermore, it was positively correlated with the biosyntheses of melanin and xylanase but dispensable for the activity of HT-toxin. [ABSTRACT FROM AUTHOR]

Monolepta hieroglyphica (Motschulsky, 1858) has been recently revised as the synonym of M. signata (Oliver, 1808) based on morphological characteristics. M. quadriguttata (Motschulsky, 1860) was synonymized with M. signata over 100 years ago. However, this synonymy has not be confirmed by molecular data. This widely distributed and abundant species shows a high variability in coloration patterns that caused numbers of synonyms. We selected color forms typical for the three nominal taxa for phylogenetic analysis. The phylogenetic, haplotype and genetic differentiation analysis of M. hieroglyphica, M. quadriguttata and M. signata were constructed to determine the relationships between them, and we also found mating behavior between them. They were intermixed together on phylogenetic trees, and M. hieroglyphica and M. quadriguttata had shared haplotype. There was no significant difference among them for genetic distance, and the synonymy of the three names based on external and genital characters can be confirmed by molecular data. [ABSTRACT FROM AUTHOR]

Rhizoctonia solani is a soil-borne pathogen with 14 anastomosis groups (AGs), and different subgroups are genetically diverse. However, the genetic factors contributing to the pathogenicity of the fungus have not been well characterized. In this study, the genome of R. solani AG1-ZJ was sequenced. As the result, a 41.57 Mb draft genome containing 12,197 putative coding genes was obtained. Comparative genomic analysis of 11 different AGs revealed conservation and unique characteristics between the AGs. Furthermore, a novel effector family containing a 68 amino acid conserved domain unique in basidiomycetous fungi was characterized. Two effectors containing the conserved domain in AG4-JY were identified, and named as RsUEB1 and RsUEB2. Furthermore, the spray-induced gene silencing strategy was used to generate a dsRNA capable of silencing the conserved domain sequence of RsUEB1 and RsUEB2. This dsRNA can significantly reduce the expression of RsUEB1 and RsUEB2 and the pathogenicity of AG4-JY on foxtail millet, maize, rice and wheat. In conclusion, this study provides significant insights into the pathogenicity mechanisms of R. solani. The identification of the conserved domain and the successful use of dsRNA silencing of the gene containing the conserved domain will offer a new strategy for controlling sheath blight in cereal crops. [ABSTRACT FROM AUTHOR]

The discovery of new targets and lead compounds is the key to developing new pesticides. The herbicidal target of drupacine has been identified as shikimate dehydrogenase (SkDH). However, the mechanism of interaction between them remains unclear. This study found that drupacine specifically binds to SkDH with a dissociation equilibrium constant (K D) of 8.88 μM and a K d value of 2.15 μM, as confirmed by surface plasmon resonance and microscale thermophoresis. Site-directed mutagenesis coupled with fluorescence quenching analysis indicated that residue THR431 was the key amino acid site for drupacine binding to SkDH. Nine compounds with the best binding ability to SkDH were identified by virtual screening from about 120,000 compounds. Among them, compound 8 showed the highest inhibition rate with values of 41.95% against SkDH, also exhibiting the strongest herbicidal activity. This research identifies a novel potential target SkDH and a candidate lead compound with high herbicidal activity for developing new herbicides. [Display omitted] • The binding ability of drupacine and SkDH was verified. • The key amino acid binding sites for drupacine and SkDH were identified. • Nine compounds targeting SkDH were identified by virtual screening. • The study validates new targets and compounds with herbicidal activity. [ABSTRACT FROM AUTHOR]

Northern corn leaf blight caused by Setosphaeria turcica is a major fungal disease responsible for significant reductions in maize yield worldwide. Eukaryotic type 2A protein phosphatase (PP2A) influences growth and virulence in a number of pathogenic fungi, but little is known about its roles in S. turcica. Here, we functionally characterized S. turcica StPP2A-C , which encodes the catalytic C subunit of StPP2A. StPP2A-C deletion slowed colony growth, conidial germination, and appressorium formation but increased conidiation, melanin biosynthesis, glycerol content, and disease lesion size on maize. These effects were associated with expression changes in genes related to calcium signaling, conidiation, laccase activity, and melanin and glycerol biosynthesis, as well as changes in intra- and extracellular laccase activity. A pull-down screen for candidate StPP2A-c interactors revealed an interaction between StPP2A-c and StLac1. Theoretical modeling and yeast two-hybrid experiments confirmed that StPP2A-c interacted specifically with the copper ion binding domain of StLac1 and that Cys267 of StPP2A-c was required for this interaction. StPP2A-C expression thus appears to promote hyphal growth and reduce pathogenicity in S. turcica , at least in part by altering melanin synthesis and laccase activity; these insights may ultimately support the development of novel strategies for biological management of S. turcica. [ABSTRACT FROM AUTHOR]

Nanotechnology can improve the stability, slow release, and effectiveness of botanical pesticides. Eleocharis dulcis peel extract (EDPE) was found to have a potent aphidicidal activity in our past study. In order to increase its properties, EDPE nanoliposomes was prepared and its aphidicidal activity was also tested. The optimal preparation process of EDPE nanoliposomes was determined by single-factor examination and Box-Behnken response surface test design as soybean lecithin: cholesterol: EDP extract mass ratio of was 5:4.1:1, medium PBS pH of 7.3, hydration temperature of 45 °C, and chloroform as the organic solvent; the encapsulation ratio of EDPE nanoliposomes prepared by using the optimal prescription was 90.67%. The scanning electron microscope and transmission electron microscopy results showed that the nanoliposomes were round or ova; and the particle size was 222.55 ± 2.59 nm; the polydispersity index was 0.11 ± 0.03; the zeta potential was − 4.78 ± 2.61 mV. The stability test showed that the EDPE nanoliposomes had good stability within 60 d. EDPE nanoliposomes showed potent aphidicidal activity, and the LC 50 were 136.59 and 83.99 mg/L for 48 and 72 h against Megoura crassicauda , respectively; and 315.78 and 154.34 mg/L against Acyrthosiphon pisum , respectively, higher than that of matrine. In addition, compared to non-capsulated EDPE, EDPE nano-liposomes improved both aphidicidal activity and the effective period. It follows then that EDPE nanoliposomes have the potential to be developed into botanical aphicides. • The Eleocharis dulcis corm-peels nanoliposomes were developed. • The encapsulation ratio of EDPE nanoliposomes was 90.67%, with good stability. • EDPE nanoliposomes had high aphicidal activity. • EDPE nanoliposomes have the potential to be developed as a botanical aphicide. [ABSTRACT FROM AUTHOR]

The active laccases of ascomycetous fungus Setosphaeria turcica were identified by Native-PAGE and ESI-MS/MS, and one of these isozymes Stlac2 was heterologous expressed to investigate the decolorization of malachite green. Setosphaeria turcica produced three active laccase isozymes: Stlac1, Stlac2, and Stlac6. Stlac2 was heterologously expressed in both eukaryotic and prokaryotic expression systems. The eukaryotic recombinant Stlac2 expressed in Pichia pastoris was inactive, and also showed a higher molecular weight than predicted because of glycosylation. The depression of laccase activity was attributable to the incorrect glycosylation at Asn97. Stlac2 expressed in Escherichia coli and the recombinant Stlac2 exhibited activity of 28.23 U/mg with 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. The highest activity was observed at pH of 4.5 and the temperature of 60 °C. The activity of recombinant Stlac2 was inhibited by 10 mM Na+, Mg2+, Ca2+, Mn2+, and increased by 10 mM of Fe3+ with a relatively activity of 315% compared with no addition. Cu2+ did not affect enzyme activity. Recombinant Stlac2 was capable of decolorizing 67.08% of 20 mg/L malachite green in 15 min without any mediators. Generally, recombinant protein of fungal laccase Stlac2 was active without glycosylation and decolorize malachite green efficiently, which has potential industrial applications. [ABSTRACT FROM AUTHOR]

Natural products are one of the important sources for the creation of new pesticides. Drupacine ((1 R ,11 S ,12 S ,13 R ,15 S)-13-methoxy-5,7,21-trioxa-19-azahexacyclo[11.7.1.02,10.04,8.011,15.015,19]henicosa-2,4(8),9-trien-12-ol), isolated from Cephalotaxus sinensis (Chinese plum-yew), is a potent herbicidal compound containing an oxo-bridged oxygen bond structure. However, its molecular target still remains unknown. In this study, the targets of drupacine in Amaranthus retroflexus were identified by combining drug affinity responsive target stability (DARTS), cellular thermal shift assay coupled with mass spectrometry (CETSA MS), RNA-seq transcriptomic, and TMT proteomic analyses. Fifty-one and sixty-eight main binding proteins were identified by DARTS and CETSA MS, respectively, including nine co-existing binding proteins. In drupacine-treated A. retroflexus seedlings we identified 1389 up-regulated genes and 442 down-regulated genes, 34 up-regulated proteins, and 194 down-regulated proteins, respectively. Combining the symptoms and the biochemical profiles, Profilin, Shikimate dehydrogenase (SkDH), and Zeta-carotene desaturase were predicted to be the drupacine potential target proteins. At the same time, drupacine was found to bind SkDH stronger by molecular docking, and its inhibition on ArSkDH increased with the treatment concentration increase. Our results suggest that the molecular target of drupacine is SkDH, a new herbicide target, which lay a foundation for the rational design of herbicides based on new targets from natural products and enrich the target resources for developing green herbicides. [Display omitted] • We have identified 51 possible protein targets of drupacine by drug affinity responsive target stability. • We identified 68 binding proteins by cellular thermal shift assay coupled with mass spectrometry. • Profilin, Shikimate dehydrogenase, and Zeta-carotene desaturase were predicted to be the drupacine potential target proteins. • Drupacine exhibited strong Shikimate dehydrogenase inhibitory activity with superior binding affinities. • This research enriches the target resources for developing green herbicides. [ABSTRACT FROM AUTHOR]

Maize ear rot is a widespread disease and the main pathogen is Fusarium verticillioides. Plant microRNAs (miRNAs) have great effects on disease resistance and it has been reported that maize miRNA participates in defense responses in maize ear rot. However, the trans-kingdom regulation of miRNAs between maize and F. verticillioides remains uncharacterized. In this study, the relationship between miRNA-like RNAs (milRNAs) of F. verticillioides and pathogenicity was investigated, followed by sRNA analysis and degradome sequencing of miRNA profiles and the target genes of maize and F. verticillioides after inoculation. It was found that the milRNA biogenesis positively regulated the pathogenicity of F. verticillioides by knocking out the gene FvDicer2 -encoded Dicer-like protein in F. verticillioides. Following inoculation with F. verticillioides , 284 known and 6571 novel miRNAs were obtained in maize, including 28 miRNAs differentially expressed at multiple time points. The target genes of maize differentially expressed miRNAs in F. verticillioides mediated multiple pathways, including autophagy and MAPK signaling pathway. Fifty-one novel F. verticillioides milRNAs were predicted to target 333 genes in maize involved in MAPK signaling pathways, plant hormone signaling transduction and plant - pathogen interaction pathways. Additionally, the miR528b-5p in maize targeted the mRNA of FvTTP which encoded a twice transmembrane protein in F. verticillioides. The FvTTP -knockout mutants displayed decreased pathogenicity and reduced synthesis of fumonisins. Thus, by interfering with the translation of FvTTP , the miR528b-5p inhibited F. verticillioides infection. These findings suggested a novel function of miR528 in resisting F. verticillioides infection. The miRNAs identified in this research and their putative target genes can be used to further elucidate the trans-kingdom functions of microRNAs in plant pathogen interaction. [ABSTRACT FROM AUTHOR]

Absolute quantitation (iTRAQ) is the latest development in the new quantitative proteomics technology for high-throughput identification and quantitation of proteins. The mechanisms underlying the 4-hydroxy-3-methoxy cinnamic acid ethyl ester treatment in Arabidopsis thaliana was investigated. Deficiency-induced changes in the protein profile of A . thaliana caused by this compound were analyzed using iTRAQ and quantitative real-time PCR. A total of 2909 proteins were quantified, of which 49 and 34 proteins were upregulated and downregulated, respectively, in the experimental plants compared with the controls. Treatment results showed that numerous proteins were involved in photosystemII, energy metabolism, and cell structure formation. Based on the upregulated and downregulated proteins, high amount of AT4G21280 protein acted on the oxygen-evolving enhancer protein 3-1, while low amount of AT1G10340 protein affected the catabolic process of the photosystemII-associated light-harvesting complex II. We selected these proteins to preliminarily verify the expression of proteins using quantitative real-time PCR to provide a reliable basis for further studies after proteomics analysis. Results show that the combined use of iTRAQ and quantitative real-time PCR provides an effective method to study proteins, leading to the determination of a new herbicide mechanism. [ABSTRACT FROM AUTHOR]

The trichomes of Arabidopsis thaliana serve as accumulation sites for heavy metals such as Cd2+, and thereby both help plants cope with heavy metal stress and detoxify the soil. These trichomes are also believed to prime plant defenses against insect herbivores in response to mechanical stimulation. Because Cd2+ in such trichomes may be beneficial for plant defenses, we hypothesized that mechanical stimulation would enhance sequestration of Cd2+ in trichomes. We quantified the distribution and concentration of Cd2+ in leaves of A. thaliana , of the glabrous mutant gl1–1 of A. thaliana , and Brassica rapa L. subsp. pekinensis (Lour.) Hanelt (Chinese cabbage) and examined how these changed following mechanical stimulation of the trichomes or leaves. Light brushing or exposure to caterpillars of Spodoptera exigua led trichomes of both A. thaliana and Chinese cabbage to accumulate Cd2+ complexes more rapidly and to a higher concentration than trichomes in unstimulated controls. Comparison to responses in leaves of gl1–1 mutants suggested that this acceleration and enhancement of Cd2+ storage requires signaling through trichomes. In wild type A. thaliana , Cd2+ was found exclusively in trichomes, whereas in gl1–1 mutants, Cd2+ was found mainly in the - mesophyll cells. Results suggest a mechanobiological pathway for improving heavy metal detoxification of soils through the action of hyperaccumulator plant leaves containing non-glandular trichomes. [Display omitted] • Mechanical stimulation of trichomes accelerated Cd2+ accumulation in trichome. • Mechanical stimulation of trichomes increased Cd2+ concentration in the leaf. • Mechanical stimulation of A. thaliana trichomes depleted Cd2+ from culture plates. • Mechanical stimulation of trichomes improved the phytoextraction capacity. [ABSTRACT FROM AUTHOR]

Northern corn leaf blight is a devastating disease caused by Setosphaeria turcica (S. turcica), leading to significant yield losses in maize. S. turcica initiates infection through a specialized structure known as the appressorium, which only forms on conducive substrates. In this study, we introduce a semi‑silicone water polyurethane resin (Si-PUD) that induces only germ tube formation from S. turcica conidia. A mixed coating of Si-PUD and polytetrafluoroethylene successfully triggers appressorium formation. Both coatings maintain optical transparency, chemical resistance, and thermal stability, which facilitate microscopic observations and the development of high-throughput systems. These coatings also demonstrate similar effects on Bipolaris maydis (B. maydis), suggesting their potential universal applicability. Utilizing coating-induced synchronous appressorium formation and proteomic analyses, we identified five genes essential for S. turcica appressorium development. Functional analyses of two zinc binuclear cluster domain-containing transcription factors, StTF1 and StTF2, revealed their critical roles in appressorium development and pathogenicity. This study not only develops a novel method for inducing appressorium formation but also lays the groundwork for rapid screening of environmentally-friendly fungicides that inhibit appressorium development., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)